https://proteopedia.org/api.php?action=feedcontributions&feedformat=atom&user=Wally+NovakProteopedia - User contributions [en]2026-09-13T04:15:57ZUser contributionsMediaWiki 1.43.8https://proteopedia.org/index.php?title=Ann_Taylor/Hemoglobin&diff=4409527Ann Taylor/Hemoglobin2026-02-09T23:41:10Z<p>Wally Novak: In a previous change I added the </StructureSection> tag, which allows the molecular viewer and scenes to work.</p>
<hr />
<div><StructureSection load='1gzx' size='350' side='right' caption="Human Hemoglobin α chain (grey and pink) β chain (green and yellow) with bound O2 [[1gzx]]" scene="Hemoglobin/1gzx/2" ><br />
==Function==<br />
'''Hemoglobin''' is an oxygen-transport protein. Hemoglobin is an allosteric protein. It is a <jmol><br />
<jmolLink><br />
<script> script /scripts/32/32/Subunits_1hho/1.spt; <br />
center visible;</script><br />
<text>tetramer</text><br />
</jmolLink><br />
</jmol> composed of two types of subunits designated α and β, with stoichiometry <scene name='Hemoglobin/Alpha2beta2/7'>α2β2</scene>. The <scene name='Hemoglobin/Foursubunits/5'>four subunits</scene> of hemoglobin sit roughly at the corners of a tetrahedron, facing each other across a <scene name='57/576710/Cavity/1'>cavity</scene> at the center of the molecule. Each of the subunits <scene name='Hemoglobin/Bbsubunitswithheme/5'>contains a heme</scene> prosthetic group. The <scene name='Hemoglobin/4heme/3'>heme molecules</scene> give hemoglobin its red color. <br />
<br />
The α and β subunits have very similar structures, despite their sequence differences. We will use a single <scene name='57/576710/A_subunit_rainbow/1'>α chain</scene> to examine the subunit structure more closely. The 6 major and 2 short α-helices that make up the structure of a Hb subunit (the "globin fold") are <scene name='57/576710/A_subunit_labelled_helices/1'>labeled A through H</scene>, which is the traditional naming scheme. The helices form an approximately-cylindrical bundle, with the heme and its central Fe atom bound in a <scene name='57/576710/Hydrophobic_pocket/1'>hydrophobic pocket</scene> (hydrophobic = grey; hydrophilic = purple). The proximal histidine (the tightest protein-Fe intraction) is often called <scene name='57/576710/His_f9/2'>His F9</scene>, since it is residue 9 on helix F (it is residue 87 in the human α chain). A second histidine is near the bound oxygen, and is referred to as the <scene name='57/576710/Distal_his/3'>distal histidine</scene>. In the deoxy state, the Fe2+ is <scene name='57/576710/Deoxy_non_planarity/2'>below the plane</scene> of the porphyrin ring. When oxygen is bound, the iron changes spin state, resulting in the iron moving <scene name='57/576710/Oxy_fe_planarity/3'>into the plane</scene> of the heme.<br />
<br />
<scene name='32/32/Cv/2'>This animation scene</scene> made by ''Alexander Berchansky'' shows the <span style="color:pink;background-color:black;font-weight:bold;">oxy (in pink)</span> and <span style="color:deepskyblue;background-color:black;font-weight:bold;">deoxy (in deepskyblue)</span> α1 heme groups were superimposed on each other, to give a local comparison at this site, a closeup around the heme O2-binding site. The heme is quite domed in the <span style="color:deepskyblue;background-color:black;font-weight:bold;">deepskyblue T-state (deoxy) form</span>, with the 5-coordinate, high-spin <span style="color:orange;background-color:black;font-weight:bold;">Fe (orange ball)</span> out of the plane. In the <span style="color:pink;background-color:black;font-weight:bold;">pink R-state form</span> a CO molecule is bound at the right <span style="color:lime;background-color:black;font-weight:bold;">(C in green</span>,<font color='red'><b>O in red</b></font>); the Fe, now 6-coordinate low-spin, has moved into the heme plane, which has flattenened. The proximal His (at left) connects the Fe to helices on the proximal side, making the Fe position sensitive to changes in the globin structure and vice versa. Remember that this scene shows a subunit in the all-unliganded versus the all-liganded states of Hb; when oxygen binds to just one subunit, then its internal structure undergoes some but not all of these changes, depending on conditions. <jmol><jmolButton><br />
<script>if (_animating); anim pause;set echo bottom left; color echo white; font echo 20 sansserif;echo Animation Paused; else; anim resume; set echo off;endif;</script><br />
<text>Toggle Animation</text><br />
</jmolButton></jmol><br />
<br />
Perhaps the most well-known disease caused by a mutation in the hemoglobin protein is sickle-cell anemia. It results from a mutation of the sixth residue in the β hemoglobin monomer from <scene name='57/576710/Glu_to_val/1'>glutamic acid to a valine</scene>. This hemoglobin variant is called 'hemoglobin S' ([[2hbs]]).<br />
<br />
<br />
==T to R transition==<br />
For hemoglobin to function as an oxygen-carrier in the blood, it must have an equilibrium between the two main states of its quaternary structure, the unliganded "deoxy" or "T state" versus the liganded "oxy" or "R state". The unliganded (deoxy) form is called the "T" (for "tense") state because it contains extra stabilizing interactions between the subunits, specifically <scene name='57/576710/Deoxy_salt_bridges/3'>ionic interactions</scene>. In the high oxygen affinity R-state conformation, these ionic interactions <scene name='57/576710/Oxy_ionic_interactions/1'>are lost</scene>, and the tetramer is described as "relaxed". In some organisms this difference is so pronounced that their Hb molecules dissociate into dimers in the oxygenated form. Structural changes that occur during this transition can illuminate how such changes result in important functional properties, such as cooperativity of oxygen binding and allosteric control by pH and anions. <br />
<br />
'''The Bohr effect''' is the increased stability of the T state due to protonation of histidine residues, especially <scene name='57/576710/Bohr_effect/2'>His 146</scene> of the beta chains. This is the C terminal residue of the beta chain. In the T state, the C terminal carboxylate group interacts with the positively charged side chain of lysine 40 of an alpha chain. When His 146 is protonated, it can also form an ionic interaction with Asp 94. This second interaction is one of several interactions which stabilizes the T state at lower pH. <br />
<br />
'''Bisphosphoglycerate (BPG)''' is a biproduct of metabolism; its presence is an indication of increased need for oxygen in the tissues. It binds in the <scene name='57/576710/Bpg_binding/1'>central cavity</scene> of hemoglobin, but only in the deoxy (T) state. The binding is due to interactions with <scene name='57/576710/Bpg_binding_residues/2'>positively charged residues</scene>. In the oxy form, this cavity is much narrower, and BPG cannot bind. <br />
<br />
<br />
----<br />
<br />
Other Species<br />
<br />
Some fish exhibit a more extreme stabilization at low pH, to the extent that the fully oxygenated species cannot be generated at atmospheric oxygen concentrations. <ref>PMID: 15117955</ref> This is due to several ionic interactions not found in the human or mammalian hemoglobins. A novel salt bridge is found between His-69 and Asp-72 of the beta chains in the T state. Furthermore, <scene name='57/576710/Asp_tyr_asn_deoxy_5/2'>Asp99β1 binds to Tyr43α2</scene> and Asn99α2 in the T state but not the R state. Additional proton binding to the T state occurs through a pair of carboxyl groups, <scene name='57/576710/Asp_asp_deoxy/2'>Asp-96α1 and Asp-101β2</scene>. These groups share a proton in the T state that is lost in the R state as the two αβ dimers rotate, pulling the carboxyl side chains apart, allowing them to both have a negative charge. Interestingly, no salt bridge is formed by His-146 at C terminus of the beta chain, in contrast to the Bohr effect seen in human hemoglobin and described above. This may be because the serine at position 93 is changed to a cysteine in Tuna, which seems to prevent this interaction rather than strengthen it.<br />
</StructureSection><br />
<br />
==References==<br />
<references/> <br />
=='''Content Donators'''==<br />
Much of this page's content originally came from the [[Hemoglobin]] page. Many thanks to ''Alexander Berchansky'' for the hemoglobin animation. To ensure stability during my class and to include some specific data we will be using in a paper discussion, this page was created.</div>Wally Novakhttps://proteopedia.org/index.php?title=Ann_Taylor/Hemoglobin&diff=4409526Ann Taylor/Hemoglobin2026-02-09T23:39:43Z<p>Wally Novak: </p>
<hr />
<div><StructureSection load='1gzx' size='350' side='right' caption="Human Hemoglobin α chain (grey and pink) β chain (green and yellow) with bound O2 [[1gzx]]" scene="Hemoglobin/1gzx/2" ><br />
==Function==<br />
'''Hemoglobin''' is an oxygen-transport protein. Hemoglobin is an [[allosteric protein]]. It is a <jmol><br />
<jmolLink><br />
<script> script /scripts/32/32/Subunits_1hho/1.spt; <br />
center visible;</script><br />
<text>tetramer</text><br />
</jmolLink><br />
</jmol> composed of two types of subunits designated α and β, with stoichiometry <scene name='Hemoglobin/Alpha2beta2/7'>α2β2</scene>. The <scene name='Hemoglobin/Foursubunits/5'>four subunits</scene> of hemoglobin sit roughly at the corners of a tetrahedron, facing each other across a <scene name='57/576710/Cavity/1'>cavity</scene> at the center of the molecule. Each of the subunits <scene name='Hemoglobin/Bbsubunitswithheme/5'>contains a heme</scene> prosthetic group. The <scene name='Hemoglobin/4heme/3'>heme molecules</scene> give hemoglobin its red color. <br />
<br />
The α and β subunits have very similar structures, despite their sequence differences. We will use a single <scene name='57/576710/A_subunit_rainbow/1'>α chain</scene> to examine the subunit structure more closely. The 6 major and 2 short α-helices that make up the structure of a Hb subunit (the "globin fold") are <scene name='57/576710/A_subunit_labelled_helices/1'>labeled A through H</scene>, which is the traditional naming scheme. The helices form an approximately-cylindrical bundle, with the heme and its central Fe atom bound in a <scene name='57/576710/Hydrophobic_pocket/1'>hydrophobic pocket</scene> (hydrophobic = grey; hydrophilic = purple). The proximal histidine (the tightest protein-Fe intraction) is often called <scene name='57/576710/His_f9/2'>His F9</scene>, since it is residue 9 on helix F (it is residue 87 in the human α chain). A second histidine is near the bound oxygen, and is referred to as the <scene name='57/576710/Distal_his/3'>distal histidine</scene>. In the deoxy state, the Fe2+ is <scene name='57/576710/Deoxy_non_planarity/2'>below the plane</scene> of the porphyrin ring. When oxygen is bound, the iron changes spin state, resulting in the iron moving <scene name='57/576710/Oxy_fe_planarity/3'>into the plane</scene> of the heme.<br />
<br />
<scene name='32/32/Cv/2'>This animation scene</scene> made by ''Alexander Berchansky'' shows the <span style="color:pink;background-color:black;font-weight:bold;">oxy (in pink)</span> and <span style="color:deepskyblue;background-color:black;font-weight:bold;">deoxy (in deepskyblue)</span> α1 heme groups were superimposed on each other, to give a local comparison at this site, a closeup around the heme O2-binding site. The heme is quite domed in the <span style="color:deepskyblue;background-color:black;font-weight:bold;">deepskyblue T-state (deoxy) form</span>, with the 5-coordinate, high-spin <span style="color:orange;background-color:black;font-weight:bold;">Fe (orange ball)</span> out of the plane. In the <span style="color:pink;background-color:black;font-weight:bold;">pink R-state form</span> a CO molecule is bound at the right <span style="color:lime;background-color:black;font-weight:bold;">(C in green</span>,<font color='red'><b>O in red</b></font>); the Fe, now 6-coordinate low-spin, has moved into the heme plane, which has flattenened. The proximal His (at left) connects the Fe to helices on the proximal side, making the Fe position sensitive to changes in the globin structure and vice versa. Remember that this scene shows a subunit in the all-unliganded versus the all-liganded states of Hb; when oxygen binds to just one subunit, then its internal structure undergoes some but not all of these changes, depending on conditions. <jmol><jmolButton><br />
<script>if (_animating); anim pause;set echo bottom left; color echo white; font echo 20 sansserif;echo Animation Paused; else; anim resume; set echo off;endif;</script><br />
<text>Toggle Animation</text><br />
</jmolButton></jmol><br />
<br />
Perhaps the most well-known disease caused by a mutation in the hemoglobin protein is sickle-cell anemia. It results from a mutation of the sixth residue in the β hemoglobin monomer from <scene name='57/576710/Glu_to_val/1'>glutamic acid to a valine</scene>. This hemoglobin variant is called 'hemoglobin S' ([[2hbs]]).<br />
<br />
<br />
==T to R transition==<br />
For hemoglobin to function as an oxygen-carrier in the blood, it must have an equilibrium between the two main states of its quaternary structure, the unliganded "deoxy" or "T state" versus the liganded "oxy" or "R state". The unliganded (deoxy) form is called the "T" (for "tense") state because it contains extra stabilizing interactions between the subunits, specifically <scene name='57/576710/Deoxy_salt_bridges/3'>ionic interactions</scene>. In the high oxygen affinity R-state conformation, these ionic interactions <scene name='57/576710/Oxy_ionic_interactions/1'>are lost</scene>, and the tetramer is described as "relaxed". In some organisms this difference is so pronounced that their Hb molecules dissociate into dimers in the oxygenated form. Structural changes that occur during this transition can illuminate how such changes result in important functional properties, such as cooperativity of oxygen binding and allosteric control by pH and anions. <br />
<br />
'''The Bohr effect''' is the increased stability of the T state due to protonation of histidine residues, especially <scene name='57/576710/Bohr_effect/2'>His 146</scene> of the beta chains. This is the C terminal residue of the beta chain. In the T state, the C terminal carboxylate group interacts with the positively charged side chain of lysine 40 of an alpha chain. When His 146 is protonated, it can also form an ionic interaction with Asp 94. This second interaction is one of several interactions which stabilizes the T state at lower pH. <br />
<br />
'''Bisphosphoglycerate (BPG)''' is a biproduct of metabolism; its presence is an indication of increased need for oxygen in the tissues. It binds in the <scene name='57/576710/Bpg_binding/1'>central cavity</scene> of hemoglobin, but only in the deoxy (T) state. The binding is due to interactions with <scene name='57/576710/Bpg_binding_residues/2'>positively charged residues</scene>. In the oxy form, this cavity is much narrower, and BPG cannot bind. <br />
<br />
<br />
----<br />
<br />
Other Species<br />
<br />
Some fish exhibit a more extreme stabilization at low pH, to the extent that the fully oxygenated species cannot be generated at atmospheric oxygen concentrations. <ref>PMID: 15117955</ref> This is due to several ionic interactions not found in the human or mammalian hemoglobins. A novel salt bridge is found between His-69 and Asp-72 of the beta chains in the T state. Furthermore, <scene name='57/576710/Asp_tyr_asn_deoxy_5/2'>Asp99β1 binds to Tyr43α2</scene> and Asn99α2 in the T state but not the R state. Additional proton binding to the T state occurs through a pair of carboxyl groups, <scene name='57/576710/Asp_asp_deoxy/2'>Asp-96α1 and Asp-101β2</scene>. These groups share a proton in the T state that is lost in the R state as the two αβ dimers rotate, pulling the carboxyl side chains apart, allowing them to both have a negative charge. Interestingly, no salt bridge is formed by His-146 at C terminus of the beta chain, in contrast to the Bohr effect seen in human hemoglobin and described above. This may be because the serine at position 93 is changed to a cysteine in Tuna, which seems to prevent this interaction rather than strengthen it.<br />
</StructureSection><br />
<br />
==References==<br />
<references/> <br />
=='''Content Donators'''==<br />
Much of this page's content originally came from the [[Hemoglobin]] page. Many thanks to ''Alexander Berchansky'' for the hemoglobin animation. To ensure stability during my class and to include some specific data we will be using in a paper discussion, this page was created.</div>Wally Novakhttps://proteopedia.org/index.php?title=Ann_Taylor/Hemoglobin&diff=4409525Ann Taylor/Hemoglobin2026-02-09T23:38:23Z<p>Wally Novak: </p>
<hr />
<div><StructureSection load='1gzx' size='350' side='right' caption="Human Hemoglobin α chain (grey and pink) β chain (green and yellow) with bound O2 [[1gzx]]" scene="Hemoglobin/1gzx/2" ><br />
==Function==<br />
'''Hemoglobin''' is an oxygen-transport protein. Hemoglobin is an [[allosteric protein]]. It is a tetramer composed of two types of subunits designated α and β, with stoichiometry <scene name='Hemoglobin/Alpha2beta2/7'>α2β2</scene>. The <scene name='Hemoglobin/Foursubunits/5'>four subunits</scene> of hemoglobin sit roughly at the corners of a tetrahedron, facing each other across a <scene name='57/576710/Cavity/1'>cavity</scene> at the center of the molecule. Each of the subunits <scene name='Hemoglobin/Bbsubunitswithheme/5'>contains a heme</scene> prosthetic group. The <scene name='Hemoglobin/4heme/3'>heme molecules</scene> give hemoglobin its red color. <br />
<br />
The α and β subunits have very similar structures, despite their sequence differences. We will use a single <scene name='57/576710/A_subunit_rainbow/1'>α chain</scene> to examine the subunit structure more closely. The 6 major and 2 short α-helices that make up the structure of a Hb subunit (the "globin fold") are <scene name='57/576710/A_subunit_labelled_helices/1'>labeled A through H</scene>, which is the traditional naming scheme. The helices form an approximately-cylindrical bundle, with the heme and its central Fe atom bound in a <scene name='57/576710/Hydrophobic_pocket/1'>hydrophobic pocket</scene> (hydrophobic = grey; hydrophilic = purple). The proximal histidine (the tightest protein-Fe intraction) is often called <scene name='57/576710/His_f9/2'>His F9</scene>, since it is residue 9 on helix F (it is residue 87 in the human α chain). A second histidine is near the bound oxygen, and is referred to as the <scene name='57/576710/Distal_his/3'>distal histidine</scene>. In the deoxy state, the Fe2+ is <scene name='57/576710/Deoxy_non_planarity/2'>below the plane</scene> of the porphyrin ring. When oxygen is bound, the iron changes spin state, resulting in the iron moving <scene name='57/576710/Oxy_fe_planarity/3'>into the plane</scene> of the heme.<br />
<br />
<scene name='32/32/Cv/2'>This animation scene</scene> made by ''Alexander Berchansky'' shows the <span style="color:pink;background-color:black;font-weight:bold;">oxy (in pink)</span> and <span style="color:deepskyblue;background-color:black;font-weight:bold;">deoxy (in deepskyblue)</span> α1 heme groups were superimposed on each other, to give a local comparison at this site, a closeup around the heme O2-binding site. The heme is quite domed in the <span style="color:deepskyblue;background-color:black;font-weight:bold;">deepskyblue T-state (deoxy) form</span>, with the 5-coordinate, high-spin <span style="color:orange;background-color:black;font-weight:bold;">Fe (orange ball)</span> out of the plane. In the <span style="color:pink;background-color:black;font-weight:bold;">pink R-state form</span> a CO molecule is bound at the right <span style="color:lime;background-color:black;font-weight:bold;">(C in green</span>,<font color='red'><b>O in red</b></font>); the Fe, now 6-coordinate low-spin, has moved into the heme plane, which has flattenened. The proximal His (at left) connects the Fe to helices on the proximal side, making the Fe position sensitive to changes in the globin structure and vice versa. Remember that this scene shows a subunit in the all-unliganded versus the all-liganded states of Hb; when oxygen binds to just one subunit, then its internal structure undergoes some but not all of these changes, depending on conditions. <jmol><jmolButton><br />
<script>if (_animating); anim pause;set echo bottom left; color echo white; font echo 20 sansserif;echo Animation Paused; else; anim resume; set echo off;endif;</script><br />
<text>Toggle Animation</text><br />
</jmolButton></jmol><br />
<br />
Perhaps the most well-known disease caused by a mutation in the hemoglobin protein is sickle-cell anemia. It results from a mutation of the sixth residue in the β hemoglobin monomer from <scene name='57/576710/Glu_to_val/1'>glutamic acid to a valine</scene>. This hemoglobin variant is called 'hemoglobin S' ([[2hbs]]).<br />
<br />
<br />
==T to R transition==<br />
For hemoglobin to function as an oxygen-carrier in the blood, it must have an equilibrium between the two main states of its quaternary structure, the unliganded "deoxy" or "T state" versus the liganded "oxy" or "R state". The unliganded (deoxy) form is called the "T" (for "tense") state because it contains extra stabilizing interactions between the subunits, specifically <scene name='57/576710/Deoxy_salt_bridges/3'>ionic interactions</scene>. In the high oxygen affinity R-state conformation, these ionic interactions <scene name='57/576710/Oxy_ionic_interactions/1'>are lost</scene>, and the tetramer is described as "relaxed". In some organisms this difference is so pronounced that their Hb molecules dissociate into dimers in the oxygenated form. Structural changes that occur during this transition can illuminate how such changes result in important functional properties, such as cooperativity of oxygen binding and allosteric control by pH and anions. <br />
<br />
'''The Bohr effect''' is the increased stability of the T state due to protonation of histidine residues, especially <scene name='57/576710/Bohr_effect/2'>His 146</scene> of the beta chains. This is the C terminal residue of the beta chain. In the T state, the C terminal carboxylate group interacts with the positively charged side chain of lysine 40 of an alpha chain. When His 146 is protonated, it can also form an ionic interaction with Asp 94. This second interaction is one of several interactions which stabilizes the T state at lower pH. <br />
<br />
'''Bisphosphoglycerate (BPG)''' is a biproduct of metabolism; its presence is an indication of increased need for oxygen in the tissues. It binds in the <scene name='57/576710/Bpg_binding/1'>central cavity</scene> of hemoglobin, but only in the deoxy (T) state. The binding is due to interactions with <scene name='57/576710/Bpg_binding_residues/2'>positively charged residues</scene>. In the oxy form, this cavity is much narrower, and BPG cannot bind. <br />
<br />
<br />
----<br />
<br />
Other Species<br />
<br />
Some fish exhibit a more extreme stabilization at low pH, to the extent that the fully oxygenated species cannot be generated at atmospheric oxygen concentrations. <ref>PMID: 15117955</ref> This is due to several ionic interactions not found in the human or mammalian hemoglobins. A novel salt bridge is found between His-69 and Asp-72 of the beta chains in the T state. Furthermore, <scene name='57/576710/Asp_tyr_asn_deoxy_5/2'>Asp99β1 binds to Tyr43α2</scene> and Asn99α2 in the T state but not the R state. Additional proton binding to the T state occurs through a pair of carboxyl groups, <scene name='57/576710/Asp_asp_deoxy/2'>Asp-96α1 and Asp-101β2</scene>. These groups share a proton in the T state that is lost in the R state as the two αβ dimers rotate, pulling the carboxyl side chains apart, allowing them to both have a negative charge. Interestingly, no salt bridge is formed by His-146 at C terminus of the beta chain, in contrast to the Bohr effect seen in human hemoglobin and described above. This may be because the serine at position 93 is changed to a cysteine in Tuna, which seems to prevent this interaction rather than strengthen it.<br />
</StructureSection><br />
<br />
==References==<br />
<references/> <br />
=='''Content Donators'''==<br />
Much of this page's content originally came from the [[Hemoglobin]] page. Many thanks to ''Alexander Berchansky'' for the hemoglobin animation. To ensure stability during my class and to include some specific data we will be using in a paper discussion, this page was created.</div>Wally Novakhttps://proteopedia.org/index.php?title=Ann_Taylor/Hemoglobin&diff=4409524Ann Taylor/Hemoglobin2026-02-09T23:37:40Z<p>Wally Novak: </p>
<hr />
<div><StructureSection load='1gzx' size='350' side='right' caption="Human Hemoglobin α chain (grey and pink) β chain (green and yellow) with bound O2 [[1gzx]]" scene="Hemoglobin/1gzx/2" ><br />
==Function==<br />
'''Hemoglobin''' is an oxygen-transport protein. Hemoglobin is an [[allosteric protein]]. It is a <br />
tetramer composed of two types of subunits designated α and β, with stoichiometry <scene name='Hemoglobin/Alpha2beta2/7'>α2β2</scene>. The <scene name='Hemoglobin/Foursubunits/5'>four subunits</scene> of hemoglobin sit roughly at the corners of a tetrahedron, facing each other across a <scene name='57/576710/Cavity/1'>cavity</scene> at the center of the molecule. Each of the subunits <scene name='Hemoglobin/Bbsubunitswithheme/5'>contains a heme</scene> prosthetic group. The <scene name='Hemoglobin/4heme/3'>heme molecules</scene> give hemoglobin its red color. <br />
<br />
The α and β subunits have very similar structures, despite their sequence differences. We will use a single <scene name='57/576710/A_subunit_rainbow/1'>α chain</scene> to examine the subunit structure more closely. The 6 major and 2 short α-helices that make up the structure of a Hb subunit (the "globin fold") are <scene name='57/576710/A_subunit_labelled_helices/1'>labeled A through H</scene>, which is the traditional naming scheme. The helices form an approximately-cylindrical bundle, with the heme and its central Fe atom bound in a <scene name='57/576710/Hydrophobic_pocket/1'>hydrophobic pocket</scene> (hydrophobic = grey; hydrophilic = purple). The proximal histidine (the tightest protein-Fe intraction) is often called <scene name='57/576710/His_f9/2'>His F9</scene>, since it is residue 9 on helix F (it is residue 87 in the human α chain). A second histidine is near the bound oxygen, and is referred to as the <scene name='57/576710/Distal_his/3'>distal histidine</scene>. In the deoxy state, the Fe2+ is <scene name='57/576710/Deoxy_non_planarity/2'>below the plane</scene> of the porphyrin ring. When oxygen is bound, the iron changes spin state, resulting in the iron moving <scene name='57/576710/Oxy_fe_planarity/3'>into the plane</scene> of the heme.<br />
<br />
<scene name='32/32/Cv/2'>This animation scene</scene> made by ''Alexander Berchansky'' shows the <span style="color:pink;background-color:black;font-weight:bold;">oxy (in pink)</span> and <span style="color:deepskyblue;background-color:black;font-weight:bold;">deoxy (in deepskyblue)</span> α1 heme groups were superimposed on each other, to give a local comparison at this site, a closeup around the heme O2-binding site. The heme is quite domed in the <span style="color:deepskyblue;background-color:black;font-weight:bold;">deepskyblue T-state (deoxy) form</span>, with the 5-coordinate, high-spin <span style="color:orange;background-color:black;font-weight:bold;">Fe (orange ball)</span> out of the plane. In the <span style="color:pink;background-color:black;font-weight:bold;">pink R-state form</span> a CO molecule is bound at the right <span style="color:lime;background-color:black;font-weight:bold;">(C in green</span>,<font color='red'><b>O in red</b></font>); the Fe, now 6-coordinate low-spin, has moved into the heme plane, which has flattenened. The proximal His (at left) connects the Fe to helices on the proximal side, making the Fe position sensitive to changes in the globin structure and vice versa. Remember that this scene shows a subunit in the all-unliganded versus the all-liganded states of Hb; when oxygen binds to just one subunit, then its internal structure undergoes some but not all of these changes, depending on conditions. <jmol><jmolButton><br />
<script>if (_animating); anim pause;set echo bottom left; color echo white; font echo 20 sansserif;echo Animation Paused; else; anim resume; set echo off;endif;</script><br />
<text>Toggle Animation</text><br />
</jmolButton></jmol><br />
<br />
Perhaps the most well-known disease caused by a mutation in the hemoglobin protein is sickle-cell anemia. It results from a mutation of the sixth residue in the β hemoglobin monomer from <scene name='57/576710/Glu_to_val/1'>glutamic acid to a valine</scene>. This hemoglobin variant is called 'hemoglobin S' ([[2hbs]]).<br />
<br />
<br />
==T to R transition==<br />
For hemoglobin to function as an oxygen-carrier in the blood, it must have an equilibrium between the two main states of its quaternary structure, the unliganded "deoxy" or "T state" versus the liganded "oxy" or "R state". The unliganded (deoxy) form is called the "T" (for "tense") state because it contains extra stabilizing interactions between the subunits, specifically <scene name='57/576710/Deoxy_salt_bridges/3'>ionic interactions</scene>. In the high oxygen affinity R-state conformation, these ionic interactions <scene name='57/576710/Oxy_ionic_interactions/1'>are lost</scene>, and the tetramer is described as "relaxed". In some organisms this difference is so pronounced that their Hb molecules dissociate into dimers in the oxygenated form. Structural changes that occur during this transition can illuminate how such changes result in important functional properties, such as cooperativity of oxygen binding and allosteric control by pH and anions. <br />
<br />
'''The Bohr effect''' is the increased stability of the T state due to protonation of histidine residues, especially <scene name='57/576710/Bohr_effect/2'>His 146</scene> of the beta chains. This is the C terminal residue of the beta chain. In the T state, the C terminal carboxylate group interacts with the positively charged side chain of lysine 40 of an alpha chain. When His 146 is protonated, it can also form an ionic interaction with Asp 94. This second interaction is one of several interactions which stabilizes the T state at lower pH. <br />
<br />
'''Bisphosphoglycerate (BPG)''' is a biproduct of metabolism; its presence is an indication of increased need for oxygen in the tissues. It binds in the <scene name='57/576710/Bpg_binding/1'>central cavity</scene> of hemoglobin, but only in the deoxy (T) state. The binding is due to interactions with <scene name='57/576710/Bpg_binding_residues/2'>positively charged residues</scene>. In the oxy form, this cavity is much narrower, and BPG cannot bind. <br />
<br />
<br />
----<br />
<br />
Other Species<br />
<br />
Some fish exhibit a more extreme stabilization at low pH, to the extent that the fully oxygenated species cannot be generated at atmospheric oxygen concentrations. <ref>PMID: 15117955</ref> This is due to several ionic interactions not found in the human or mammalian hemoglobins. A novel salt bridge is found between His-69 and Asp-72 of the beta chains in the T state. Furthermore, <scene name='57/576710/Asp_tyr_asn_deoxy_5/2'>Asp99β1 binds to Tyr43α2</scene> and Asn99α2 in the T state but not the R state. Additional proton binding to the T state occurs through a pair of carboxyl groups, <scene name='57/576710/Asp_asp_deoxy/2'>Asp-96α1 and Asp-101β2</scene>. These groups share a proton in the T state that is lost in the R state as the two αβ dimers rotate, pulling the carboxyl side chains apart, allowing them to both have a negative charge. Interestingly, no salt bridge is formed by His-146 at C terminus of the beta chain, in contrast to the Bohr effect seen in human hemoglobin and described above. This may be because the serine at position 93 is changed to a cysteine in Tuna, which seems to prevent this interaction rather than strengthen it.<br />
</StructureSection><br />
<br />
==References==<br />
<references/> <br />
=='''Content Donators'''==<br />
Much of this page's content originally came from the [[Hemoglobin]] page. Many thanks to ''Alexander Berchansky'' for the hemoglobin animation. To ensure stability during my class and to include some specific data we will be using in a paper discussion, this page was created.</div>Wally Novakhttps://proteopedia.org/index.php?title=Category:Atomic_Geometry&diff=3313255Category:Atomic Geometry2020-11-09T16:16:58Z<p>Wally Novak: </p>
<hr />
<div>Atomic Geometry is one of twelve overarching themes in the Biomolecular Visualization (BioMolViz) Framework. The Framework identifies learning goals to assist in developing biomolecular visualization literacy. More information about the BioMolViz project can be found at: https://biomolviz.org/, with additional information [http://proteopedia.org/wiki/index.php/Kristen_Procko/BioMolViz here].<br />
<br />
<br />
<br />
'''Overarching theme'''<br />
<br />
Atomic Geometry (AG): three‐atom and four‐atom dihedral/torsion angles, metal size and metal‐ligand geometries, steric clashes.<br />
<br />
<br />
'''Learning Goals:'''<br />
<br />
'''AG1. '''Students can describe the ideal geometry for a given atom within a molecule and deviations from the ideal geometry due to neighboring interactions.<br />
<br />
'''AG2.''' Students can compare and contrast different structural conformations with regard to energy, addition of substituents, and impact on structure/function of a macromolecule.<br />
<br />
'''AG3.''' Students can describe the effect of dihedral/torsion angles on macromolecular structure.</div>Wally Novakhttps://proteopedia.org/index.php?title=User:Wally_Novak/Sandbox2_RNAPolII&diff=3288336User:Wally Novak/Sandbox2 RNAPolII2020-09-08T13:15:35Z<p>Wally Novak: </p>
<hr />
<div><StructureSection load='1i6h' size='340' side='right' caption='Yeast RNA Polymerase II complex with RNA (PDB code [[1i6h]])'><br />
== Introduction ==<br />
[[ Image:Label RNA pol II (1).png|150px|right|thumb| RNAP II transcription process.]]<br />
<br />
<br />
'''RNA polymerase II''' (RNAP II) is an enzyme that transcribes protein-encoding genes, and it therefore is responsible for the synthesis of mRNA. There are three RNA polymerase enzymes found in eukaryotic nuclei but RNAP II is the most studied. RNAP II is a 550 kDa multi-protein complex that includes 12 subunits. Several transcription factors are used to bind promoters upstream of the start site and are necessary for joining RNAP II and DNA. Bound RNAP II transcribes DNA into a strand of messenger RNA. Messenger RNA (mRNA) is a single stranded RNA molecule that is complementary to the template strand of DNA. The mRNA stand transports genetic information from DNA to the ribosome, where it is used to specify the amino acid sequence for the production of proteins. <br />
<br />
== Structural Components ==<br />
<br />
This section will briefly discuss the chief structural components involved in the mechanism. <br />
<br />
[[ Image:RnapII struct features.jpg|300px|left|thumb| The clamp (magenta), wall (navy blue), rudder (red), bridge (orange), RNA (light blue), and DNA (blue) are depicted. See below for PDB's and residue numbers.]]<br />
<br />
To begin, the <scene name='82/824648/Clamp/5'>clamp</scene> swings to trap the DNA in the cleft. Further along, the <scene name='82/824648/Wall/2'>wall</scene> sends the DNA template through the cleft in approximately a 90° turn. Both the clamp and wall are parts of the Rpb2 subunit. Further along in the process, the <scene name='82/824648/Rudder/3'>rudder</scene> separates the newly synthesized RNA strand from the DNA template. The DNA reforms into a double helix as it leaves RNA pol II. <br />
<br />
Other components of RNA pol II include the following:<br />
The jaw is the opening through which DNA enters. The funnel is what the NTP’s travel through to be incorporated into the growing RNA strand, and the pore is the end of the funnel. The <scene name='82/824648/Bridge/6'>bridge</scene> is an Rpb1 segment that translocates the DNA-RNA complex at the end of each cycle of catalysis. <scene name='82/824648/Magnesium/3'>Magnesium</scene> is located within the active site and functions as the catalyst. <br />
<br />
== Transcription ==<br />
<br />
Transcription can largely be divided into three sections: initiation, elongation, and termination. In the process of initiation, RNAP II recruits several general transcription factors (GTFs) to bind to the promoter region of the DNA, and this eventually forms the preinitiation complex (PIC). The DNA enters RNAP II through the clamp, and then it is unwound, creating a transcription bubble. With the DNA unwound, the active site of RNAP II catalyzes the synthesis of the first few RNA bonds. Once the carboxy-terminal domain (CTD) becomes phosphorylated, the clamp undergoes a conformation change to effectively trap the DNA, and a few of the GTFs dissociate, which changes complex to the Elongator complex. <br />
<br />
After initiation, the process of elongation begins with the entry of NTPs. These NTPs largely enter through the funnel, and this tends to be a slow process because the funnel is only 12 Å in diameter, meaning that only one NTP can go through at a time. Once they have passed through the funnel, the NTPs enter <scene name='86/860989/Active_site_wn_test/1'>active site</scene> which contains an Mg ion that is bound by three aspartate residues at positions D739, D741, and D743. If the NTP is complementary to the DNA strand, it is loaded onto the insertion site next to the RNA and held in place by the bridge. While in this insertion site, the metal ion and aspartate residues catalyze the reaction that forms a phosphodiester bond between the 3’ end of the RNA and the 5’ end of the NTP. This step then leads into a translocation step. To make room for the next NTP, there is a Brownian ratchet mechanism in which the nearby trigger loop undergoes a conformation change that causes the bridge to move to the next transition site. During the bridge’s transition, the DNA-RNA hybrid is partially help in place by the -amanitin. Once the bridge is in the new initiation site, the trigger loop returns to its original conformation, allowing the process to begin again. <br />
<br />
In the termination stage, the CTD becomes dephosphorylated, which acts as a signal to dissociate the elongation complex. Once this signal is received, the new mRNA is released from RNP II through the rudder. Before being released, its 3’ end is polyadenylated. The DNA is brought back together at the other end of the transcription bubble, returning it to its original double-stranded form. Finally, RNAP II and the remaining GTFs dissociate from the DNA. <br />
== α-Amanitin ==<br />
<br />
α-Amanitin is a bicyclic octapeptide that adheres tightly with RNAP II, which blocks the elongation steps. α-amanitin binds in the funnel and interacts with the bridge helix and adjacent Rpb1, but it does not inhibit the RNA pol II’s interaction with NTP. Instead, α-amanitin likely challenges the bridge’s conformational change that is necessary for the purposed RNAP translocation step. α-Amanitin, found in the poisonous mushroom death cap, leads to death after several days. This time frame aligns with the rate at which mRNA’s and proteins turnover. <br />
<br />
[[Image:Alpha-amanitin structure (1).png|300px|right|thumb| The chemical structure of α-amanitin.]]<br />
<br />
<br />
== General Transcription Factors ==<br />
<br />
In both eukaryotes and prokaryotes, the basic mechanism for initiating transcription is the same: protein factors selectively bind to promoter regions on DNA. Prokaryotes use sigma factors while eukaryotes use a complex of 6 GTFs. These GTFs are all named similarly and begin with TF, for transcription factor, followed by the Roman numeral II since they are involved in transcription by RNAP II. The combination of all the transcription factors bound to the DNA promoter region, in complex with RNAP II, is called the PIC. The formation of the PIC occurs in an ordered pathway, beginning with the TATA box which is a promoter region on DNA at position -27.<br />
<br />
Process of PIC formation:<br />
<br />
1. <scene name='82/824648/Tfiid-tbp/3'>TFIID</scene> contains a subunit named the TATA-binding protein (TBP), which recognizes and binds to the TATA box on the DNA promoter.<br />
<br />
2. <scene name='82/824648/Tfiib/3'>TFIIB</scene> and <scene name='82/824648/Tfiia/3'>TFIIA</scene> and interact with TBP and are recruted to the promoter.<br />
<br />
3. <scene name='82/824648/Tfiif/5'>TFIIF</scene> binds directly to RNAP II and escorts it to the promoter while TFIIB helps the complex bind correctly. <br />
<br />
4. <scene name='82/824648/Tfiie/4'>TFIIE</scene> and <scene name='82/824648/Tfiih/3'>TFIIH</scene> are sequentually recruited which completes the <scene name='82/824648/Pic/3'>PIC</scene>. <br />
<br />
<br />
<br />
Once the <scene name='82/824648/Pic/3'>PIC</scene> is formed, <scene name='82/824648/Rnap_ii/1'>RNAP II</scene> initiates RNA synthesis and produces a short transcript. When RNAP II becomes phosphorylated, it releases some of the GTFs from the complex and moves away from the promoter. TFIID stays bound to the promoter and can reinitiate transcription. The transcription factors are replaced by a new six-protein complex call the Elongator. TFIIF and TFIIH both remain associated with RNAP II during elongation. <br />
<br />
<br />
<br />
</StructureSection><br />
== References ==<br />
Bushnell, D. A.; Westover, K. D.; Davis, R. E.; Kornberg, R. D. Structural Basis of Transcription: An RNA Polymerase II-TFIIB Cocrystal at 4.5 Angstroms. Science. 2004, 303, 983-988<br />
<br />
Brueckner, F. and Cramer, P. Structural Basis of Transcription Inhibition by -amanitin and Implications for RNA Polymerase II Translocation. Nature Structure and Molecular Biology. 2008, 15, 811-818.<br />
<br />
Cramer, P.; Bushnell, D. A.; Kornberg, R. D. Structural Basis of Transcription: RNA Polymerase II at 2.8 Ångstrom Resolution. Science. 2001, 292, 1863-1876<br />
<br />
Evans, D. A.; Fitch, D. M.; Smith, T. E.; Cee, V. J. Application of Complex Aldol Reactions to the Total Synthesis of Phorboxazole B. J. Am. Chem. Soc. 2000, 122, 10033-10046.<br />
<br />
Gnatt, A. L.; Cramer, P; Fu, J.; Bushnell, D. A.; and Kornberg, R. D. Structural Basis of Transcription: An RNA Polymerase II Elongation Complex at 3.3 Å Resolution. Science. 2001, 292, 1876-1882 1i6h<br />
<br />
Hahn, S. Structure and Mechanism of the RNA Polymerase II Transcription Machinery. Nature Structure and Molecular Biology. 2004, 11, 394-403. <br />
<br />
He, Yuan, et al. Near-atomic resolution visualization of human transcription promoter opening. Nature 533.7603. 2016.<br />
<br />
Nudler, E. RNA Polymerase Active Center: The Molecular Engine of Transcription. Annu. Rev. Biochem. 2009, 78, 335-361.<br />
<br />
Orphanides, George, Thierry Lagrange, and Danny Reinberg. The general transcription factors of RNA polymerase II. Genes & development 10.21. 1996. 2657-2683<br />
<br />
Shah, N. et. al. Tyrosine-1 of RNA Polymerase II CTD Controls Global Termination of Gene Transcription in Mammals. Molecular Cell. 2018, 69, 48-61.<br />
<br />
Uzman, A.; Voet, D. Student companion Fundamentals of biochemistry: life at the molecular level, 4th ed., Donald Voet, Judith G. Voet, Charlotte W. Pratt; John Wiley & amp; Sons, 2012.<br />
<br />
Xu, J.; Lahiri, I.; Wang, W.; Wier, A.; Cianfrocco, M. A.; Chong, J.; Hare, A. A.; Dervan, P. B.; DiMaio, F.; Leschziner, A. E.; Wang, D. Structural Basis for the Initiation of Eukaryotic Transcription-coupled DNA Repair. Nature. 2017. 551, 653-657 5vvr<br />
<br />
Xin, L.; Bushnell, D. A.; and Kornburg, R. D. RNA Polymerase II Transcription: Structure and Mechanism. Biochemica et Biophysica Acta. 2013, 1829, 2-8.<br />
<br />
Yan, C., Dodd, T., He, Y., Tainer, J. A., Tsutakawa, S. E., & Ivanov, I. (2019). Transcription preinitiation complex structure and dynamics provide insight into genetic diseases. Nature Structural and Molecular Biology, 26(6), 397-406.<br />
<br />
Alpha-aminitin chemical structure image courtesy of https://en.wikipedia.org/wiki/Alpha-Amanitin#/media/File:Alpha-amanitin_structure.png<br />
<br />
== Notes ==<br />
From structural components:<br />
<br />
Structural overview: [PDB: 5VVR: with highlighted sections mentioned below]<br />
<br />
Bridge: Depicted: [PDB: 1I6H: 810-845.a]<br />
<br />
Wall: Depicted: [PDB: 1R5U: 853-919.b; 933-972.b]<br />
<br />
Clamp: Depicted: [PDB: 1R5U: 3-345.a; 1395-1435.a; 1158-1124.b]<br />
<br />
Rudder: Depicted: [PDB: 5VVR: 306-321.a]<br />
<br />
<br />
<br />
== Content Donators ==<br />
<br />
This page was created as a final project for the Advanced Biochemistry course at Wabash College during the Fall of 2019. This page was reviewed by Dr. Wally Novak of Wabash College.</div>Wally Novakhttps://proteopedia.org/index.php?title=User:Wally_Novak/Sandbox2_RNAPolII&diff=3288335User:Wally Novak/Sandbox2 RNAPolII2020-09-08T13:07:12Z<p>Wally Novak: </p>
<hr />
<div><br />
<StructureSection load='1i6h' size='340' side='right' caption='Yeast RNA Polymerase II complex with RNA (PDB code [[1i6h]])'><br />
== Introduction ==<br />
[[ Image:Label RNA pol II (1).png|150px|right|thumb| RNAP II transcription process.]]<br />
<br />
<br />
'''RNA polymerase II''' (RNAP II) is an enzyme that transcribes protein-encoding genes, and it therefore is responsible for the synthesis of mRNA. There are three RNA polymerase enzymes found in eukaryotic nuclei but RNAP II is the most studied. RNAP II is a 550 kDa multi-protein complex that includes 12 subunits. Several transcription factors are used to bind promoters upstream of the start site and are necessary for joining RNAP II and DNA. Bound RNAP II transcribes DNA into a strand of messenger RNA. Messenger RNA (mRNA) is a single stranded RNA molecule that is complementary to the template strand of DNA. The mRNA stand transports genetic information from DNA to the ribosome, where it is used to specify the amino acid sequence for the production of proteins. <br />
<br />
== Structural Components ==<br />
<br />
This section will briefly discuss the chief structural components involved in the mechanism. <br />
<br />
[[ Image:RnapII struct features.jpg|300px|left|thumb| The clamp (magenta), wall (navy blue), rudder (red), bridge (orange), RNA (light blue), and DNA (blue) are depicted. See below for PDB's and residue numbers.]]<br />
<br />
To begin, the <scene name='82/824648/Clamp/5'>clamp</scene> swings to trap the DNA in the cleft. Further along, the <scene name='82/824648/Wall/2'>wall</scene> sends the DNA template through the cleft in approximately a 90° turn. Both the clamp and wall are parts of the Rpb2 subunit. Further along in the process, the <scene name='82/824648/Rudder/3'>rudder</scene> separates the newly synthesized RNA strand from the DNA template. The DNA reforms into a double helix as it leaves RNA pol II. <br />
<br />
Other components of RNA pol II include the following:<br />
The jaw is the opening through which DNA enters. The funnel is what the NTP’s travel through to be incorporated into the growing RNA strand, and the pore is the end of the funnel. The <scene name='82/824648/Bridge/6'>bridge</scene> is an Rpb1 segment that translocates the DNA-RNA complex at the end of each cycle of catalysis. <scene name='82/824648/Magnesium/3'>Magnesium</scene> is located within the active site and functions as the catalyst. <br />
<br />
== Transcription ==<br />
<br />
Transcription can largely be divided into three sections: initiation, elongation, and termination. In the process of initiation, RNAP II recruits several general transcription factors (GTFs) to bind to the promoter region of the DNA, and this eventually forms the preinitiation complex (PIC). The DNA enters RNAP II through the clamp, and then it is unwound, creating a transcription bubble. With the DNA unwound, the active site of RNAP II catalyzes the synthesis of the first few RNA bonds. Once the carboxy-terminal domain (CTD) becomes phosphorylated, the clamp undergoes a conformation change to effectively trap the DNA, and a few of the GTFs dissociate, which changes complex to the Elongator complex. <br />
<br />
After initiation, the process of elongation begins with the entry of NTPs. These NTPs largely enter through the funnel, and this tends to be a slow process because the funnel is only 12 Å in diameter, meaning that only one NTP can go through at a time. Once they have passed through the funnel, the NTPs enter <scene name='82/824648/Active_site_2/1'>the active site</scene> which contains an Mg ion that is bound by three aspartate residues at positions D739, D741, and D743. If the NTP is complementary to the DNA strand, it is loaded onto the insertion site next to the RNA and held in place by the bridge. While in this insertion site, the metal ion and aspartate residues catalyze the reaction that forms a phosphodiester bond between the 3’ end of the RNA and the 5’ end of the NTP. This step then leads into a translocation step. To make room for the next NTP, there is a Brownian ratchet mechanism in which the nearby trigger loop undergoes a conformation change that causes the bridge to move to the next transition site. During the bridge’s transition, the DNA-RNA hybrid is partially help in place by the -amanitin. Once the bridge is in the new initiation site, the trigger loop returns to its original conformation, allowing the process to begin again. <br />
<br />
In the termination stage, the CTD becomes dephosphorylated, which acts as a signal to dissociate the elongation complex. Once this signal is received, the new mRNA is released from RNP II through the rudder. Before being released, its 3’ end is polyadenylated. The DNA is brought back together at the other end of the transcription bubble, returning it to its original double-stranded form. Finally, RNAP II and the remaining GTFs dissociate from the DNA. <br />
== α-Amanitin ==<br />
<br />
α-Amanitin is a bicyclic octapeptide that adheres tightly with RNAP II, which blocks the elongation steps. α-amanitin binds in the funnel and interacts with the bridge helix and adjacent Rpb1, but it does not inhibit the RNA pol II’s interaction with NTP. Instead, α-amanitin likely challenges the bridge’s conformational change that is necessary for the purposed RNAP translocation step. α-Amanitin, found in the poisonous mushroom death cap, leads to death after several days. This time frame aligns with the rate at which mRNA’s and proteins turnover. <br />
<br />
[[Image:Alpha-amanitin structure (1).png|300px|right|thumb| The chemical structure of α-amanitin.]]<br />
<br />
<br />
== General Transcription Factors ==<br />
<br />
In both eukaryotes and prokaryotes, the basic mechanism for initiating transcription is the same: protein factors selectively bind to promoter regions on DNA. Prokaryotes use sigma factors while eukaryotes use a complex of 6 GTFs. These GTFs are all named similarly and begin with TF, for transcription factor, followed by the Roman numeral II since they are involved in transcription by RNAP II. The combination of all the transcription factors bound to the DNA promoter region, in complex with RNAP II, is called the PIC. The formation of the PIC occurs in an ordered pathway, beginning with the TATA box which is a promoter region on DNA at position -27.<br />
<br />
Process of PIC formation:<br />
<br />
1. <scene name='82/824648/Tfiid-tbp/3'>TFIID</scene> contains a subunit named the TATA-binding protein (TBP), which recognizes and binds to the TATA box on the DNA promoter.<br />
<br />
2. <scene name='82/824648/Tfiib/3'>TFIIB</scene> and <scene name='82/824648/Tfiia/3'>TFIIA</scene> and interact with TBP and are recruted to the promoter.<br />
<br />
3. <scene name='82/824648/Tfiif/5'>TFIIF</scene> binds directly to RNAP II and escorts it to the promoter while TFIIB helps the complex bind correctly. <br />
<br />
4. <scene name='82/824648/Tfiie/4'>TFIIE</scene> and <scene name='82/824648/Tfiih/3'>TFIIH</scene> are sequentually recruited which completes the <scene name='82/824648/Pic/3'>PIC</scene>. <br />
<br />
<br />
<br />
Once the <scene name='82/824648/Pic/3'>PIC</scene> is formed, <scene name='82/824648/Rnap_ii/1'>RNAP II</scene> initiates RNA synthesis and produces a short transcript. When RNAP II becomes phosphorylated, it releases some of the GTFs from the complex and moves away from the promoter. TFIID stays bound to the promoter and can reinitiate transcription. The transcription factors are replaced by a new six-protein complex call the Elongator. TFIIF and TFIIH both remain associated with RNAP II during elongation. <br />
<br />
<br />
<br />
</StructureSection><br />
== References ==<br />
Bushnell, D. A.; Westover, K. D.; Davis, R. E.; Kornberg, R. D. Structural Basis of Transcription: An RNA Polymerase II-TFIIB Cocrystal at 4.5 Angstroms. Science. 2004, 303, 983-988<br />
<br />
Brueckner, F. and Cramer, P. Structural Basis of Transcription Inhibition by -amanitin and Implications for RNA Polymerase II Translocation. Nature Structure and Molecular Biology. 2008, 15, 811-818.<br />
<br />
Cramer, P.; Bushnell, D. A.; Kornberg, R. D. Structural Basis of Transcription: RNA Polymerase II at 2.8 Ångstrom Resolution. Science. 2001, 292, 1863-1876<br />
<br />
Evans, D. A.; Fitch, D. M.; Smith, T. E.; Cee, V. J. Application of Complex Aldol Reactions to the Total Synthesis of Phorboxazole B. J. Am. Chem. Soc. 2000, 122, 10033-10046.<br />
<br />
Gnatt, A. L.; Cramer, P; Fu, J.; Bushnell, D. A.; and Kornberg, R. D. Structural Basis of Transcription: An RNA Polymerase II Elongation Complex at 3.3 Å Resolution. Science. 2001, 292, 1876-1882 1i6h<br />
<br />
Hahn, S. Structure and Mechanism of the RNA Polymerase II Transcription Machinery. Nature Structure and Molecular Biology. 2004, 11, 394-403. <br />
<br />
He, Yuan, et al. Near-atomic resolution visualization of human transcription promoter opening. Nature 533.7603. 2016.<br />
<br />
Nudler, E. RNA Polymerase Active Center: The Molecular Engine of Transcription. Annu. Rev. Biochem. 2009, 78, 335-361.<br />
<br />
Orphanides, George, Thierry Lagrange, and Danny Reinberg. The general transcription factors of RNA polymerase II. Genes & development 10.21. 1996. 2657-2683<br />
<br />
Shah, N. et. al. Tyrosine-1 of RNA Polymerase II CTD Controls Global Termination of Gene Transcription in Mammals. Molecular Cell. 2018, 69, 48-61.<br />
<br />
Uzman, A.; Voet, D. Student companion Fundamentals of biochemistry: life at the molecular level, 4th ed., Donald Voet, Judith G. Voet, Charlotte W. Pratt; John Wiley & amp; Sons, 2012.<br />
<br />
Xu, J.; Lahiri, I.; Wang, W.; Wier, A.; Cianfrocco, M. A.; Chong, J.; Hare, A. A.; Dervan, P. B.; DiMaio, F.; Leschziner, A. E.; Wang, D. Structural Basis for the Initiation of Eukaryotic Transcription-coupled DNA Repair. Nature. 2017. 551, 653-657 5vvr<br />
<br />
Xin, L.; Bushnell, D. A.; and Kornburg, R. D. RNA Polymerase II Transcription: Structure and Mechanism. Biochemica et Biophysica Acta. 2013, 1829, 2-8.<br />
<br />
Yan, C., Dodd, T., He, Y., Tainer, J. A., Tsutakawa, S. E., & Ivanov, I. (2019). Transcription preinitiation complex structure and dynamics provide insight into genetic diseases. Nature Structural and Molecular Biology, 26(6), 397-406.<br />
<br />
Alpha-aminitin chemical structure image courtesy of https://en.wikipedia.org/wiki/Alpha-Amanitin#/media/File:Alpha-amanitin_structure.png<br />
<br />
== Notes ==<br />
From structural components:<br />
<br />
Structural overview: [PDB: 5VVR: with highlighted sections mentioned below]<br />
<br />
Bridge: Depicted: [PDB: 1I6H: 810-845.a]<br />
<br />
Wall: Depicted: [PDB: 1R5U: 853-919.b; 933-972.b]<br />
<br />
Clamp: Depicted: [PDB: 1R5U: 3-345.a; 1395-1435.a; 1158-1124.b]<br />
<br />
Rudder: Depicted: [PDB: 5VVR: 306-321.a]<br />
<br />
<br />
<br />
== Content Donators ==<br />
<br />
This page was created as a final project for the Advanced Biochemistry course at Wabash College during the Fall of 2019. This page was reviewed by Dr. Wally Novak of Wabash College.</div>Wally Novakhttps://proteopedia.org/index.php?title=User:Wally_Novak/Sandbox2_RNAPolII&diff=3288332User:Wally Novak/Sandbox2 RNAPolII2020-09-08T12:45:18Z<p>Wally Novak: New page: <StructureSection load='1i6h' size='340' side='right' caption='Yeast RNA Polymerase II complex with RNA (PDB code 1i6h)'> == Introduction == [[ Image:Label RNA pol II (1).png|150px|r...</p>
<hr />
<div><br />
<br />
<StructureSection load='1i6h' size='340' side='right' caption='Yeast RNA Polymerase II complex with RNA (PDB code [[1i6h]])'><br />
== Introduction ==<br />
[[ Image:Label RNA pol II (1).png|150px|right|thumb| RNAP II transcription process.]]<br />
<br />
<br />
'''RNA polymerase II''' (RNAP II) is an enzyme that transcribes protein-encoding genes, and it therefore is responsible for the synthesis of mRNA. There are three RNA polymerase enzymes found in eukaryotic nuclei but RNAP II is the most studied. RNAP II is a 550 kDa multi-protein complex that includes 12 subunits. Several transcription factors are used to bind promoters upstream of the start site and are necessary for joining RNAP II and DNA. Bound RNAP II transcribes DNA into a strand of messenger RNA. Messenger RNA (mRNA) is a single stranded RNA molecule that is complementary to the template strand of DNA. The mRNA stand transports genetic information from DNA to the ribosome, where it is used to specify the amino acid sequence for the production of proteins. <br />
<br />
== Structural Components ==<br />
<br />
This section will briefly discuss the chief structural components involved in the mechanism. <br />
<br />
[[ Image:RnapII struct features.jpg|300px|left|thumb| The clamp (magenta), wall (navy blue), rudder (red), bridge (orange), RNA (light blue), and DNA (blue) are depicted. See below for PDB's and residue numbers.]]<br />
<br />
To begin, the <scene name='82/824648/Clamp/5'>clamp</scene> swings to trap the DNA in the cleft. Further along, the <scene name='82/824648/Wall/2'>wall</scene> sends the DNA template through the cleft in approximately a 90° turn. Both the clamp and wall are parts of the Rpb2 subunit. Further along in the process, the <scene name='82/824648/Rudder/3'>rudder</scene> separates the newly synthesized RNA strand from the DNA template. The DNA reforms into a double helix as it leaves RNA pol II. <br />
<br />
Other components of RNA pol II include the following:<br />
The jaw is the opening through which DNA enters. The funnel is what the NTP’s travel through to be incorporated into the growing RNA strand, and the pore is the end of the funnel. The <scene name='82/824648/Bridge/6'>bridge</scene> is an Rpb1 segment that translocates the DNA-RNA complex at the end of each cycle of catalysis. <scene name='82/824648/Magnesium/3'>Magnesium</scene> is located within the active site and functions as the catalyst. <br />
<br />
== Transcription ==<br />
<br />
Transcription can largely be divided into three sections: initiation, elongation, and termination. In the process of initiation, RNAP II recruits several general transcription factors (GTFs) to bind to the promoter region of the DNA, and this eventually forms the preinitiation complex (PIC). The DNA enters RNAP II through the clamp, and then it is unwound, creating a transcription bubble. With the DNA unwound, the active site of RNAP II catalyzes the synthesis of the first few RNA bonds. Once the carboxy-terminal domain (CTD) becomes phosphorylated, the clamp undergoes a conformation change to effectively trap the DNA, and a few of the GTFs dissociate, which changes complex to the Elongator complex. <br />
<br />
After initiation, the process of elongation begins with the entry of NTPs. These NTPs largely enter through the funnel, and this tends to be a slow process because the funnel is only 12 Å in diameter, meaning that only one NTP can go through at a time. Once they have passed through the funnel, the NTPs enter <scene name='82/824648/Active_site_2/1'>the active site</scene> which contains an Mg ion that is bound by three aspartate residues at positions D739, D741, and D743. If the NTP is complementary to the DNA strand, it is loaded onto the insertion site next to the RNA and held in place by the bridge. While in this insertion site, the metal ion and aspartate residues catalyze the reaction that forms a phosphodiester bond between the 3’ end of the RNA and the 5’ end of the NTP. This step then leads into a translocation step. To make room for the next NTP, there is a Brownian ratchet mechanism in which the nearby trigger loop undergoes a conformation change that causes the bridge to move to the next transition site. During the bridge’s transition, the DNA-RNA hybrid is partially help in place by the -amanitin. Once the bridge is in the new initiation site, the trigger loop returns to its original conformation, allowing the process to begin again. <br />
<br />
In the termination stage, the CTD becomes dephosphorylated, which acts as a signal to dissociate the elongation complex. Once this signal is received, the new mRNA is released from RNP II through the rudder. Before being released, its 3’ end is polyadenylated. The DNA is brought back together at the other end of the transcription bubble, returning it to its original double-stranded form. Finally, RNAP II and the remaining GTFs dissociate from the DNA. <br />
== α-Amanitin ==<br />
<br />
α-Amanitin is a bicyclic octapeptide that adheres tightly with RNAP II, which blocks the elongation steps. α-amanitin binds in the funnel and interacts with the bridge helix and adjacent Rpb1, but it does not inhibit the RNA pol II’s interaction with NTP. Instead, α-amanitin likely challenges the bridge’s conformational change that is necessary for the purposed RNAP translocation step. α-Amanitin, found in the poisonous mushroom death cap, leads to death after several days. This time frame aligns with the rate at which mRNA’s and proteins turnover. <br />
<br />
[[Image:Alpha-amanitin structure (1).png|300px|right|thumb| The chemical structure of α-amanitin.]]<br />
<br />
<br />
== General Transcription Factors ==<br />
<br />
In both eukaryotes and prokaryotes, the basic mechanism for initiating transcription is the same: protein factors selectively bind to promoter regions on DNA. Prokaryotes use sigma factors while eukaryotes use a complex of 6 GTFs. These GTFs are all named similarly and begin with TF, for transcription factor, followed by the Roman numeral II since they are involved in transcription by RNAP II. The combination of all the transcription factors bound to the DNA promoter region, in complex with RNAP II, is called the PIC. The formation of the PIC occurs in an ordered pathway, beginning with the TATA box which is a promoter region on DNA at position -27.<br />
<br />
Process of PIC formation:<br />
<br />
1. <scene name='82/824648/Tfiid-tbp/3'>TFIID</scene> contains a subunit named the TATA-binding protein (TBP), which recognizes and binds to the TATA box on the DNA promoter.<br />
<br />
2. <scene name='82/824648/Tfiib/3'>TFIIB</scene> and <scene name='82/824648/Tfiia/3'>TFIIA</scene> and interact with TBP and are recruted to the promoter.<br />
<br />
3. <scene name='82/824648/Tfiif/5'>TFIIF</scene> binds directly to RNAP II and escorts it to the promoter while TFIIB helps the complex bind correctly. <br />
<br />
4. <scene name='82/824648/Tfiie/4'>TFIIE</scene> and <scene name='82/824648/Tfiih/3'>TFIIH</scene> are sequentually recruited which completes the <scene name='82/824648/Pic/3'>PIC</scene>. <br />
<br />
<br />
<br />
Once the <scene name='82/824648/Pic/3'>PIC</scene> is formed, <scene name='82/824648/Rnap_ii/1'>RNAP II</scene> initiates RNA synthesis and produces a short transcript. When RNAP II becomes phosphorylated, it releases some of the GTFs from the complex and moves away from the promoter. TFIID stays bound to the promoter and can reinitiate transcription. The transcription factors are replaced by a new six-protein complex call the Elongator. TFIIF and TFIIH both remain associated with RNAP II during elongation. <br />
<br />
<br />
<br />
</StructureSection><br />
== References ==<br />
Bushnell, D. A.; Westover, K. D.; Davis, R. E.; Kornberg, R. D. Structural Basis of Transcription: An RNA Polymerase II-TFIIB Cocrystal at 4.5 Angstroms. Science. 2004, 303, 983-988<br />
<br />
Brueckner, F. and Cramer, P. Structural Basis of Transcription Inhibition by -amanitin and Implications for RNA Polymerase II Translocation. Nature Structure and Molecular Biology. 2008, 15, 811-818.<br />
<br />
Cramer, P.; Bushnell, D. A.; Kornberg, R. D. Structural Basis of Transcription: RNA Polymerase II at 2.8 Ångstrom Resolution. Science. 2001, 292, 1863-1876<br />
<br />
Evans, D. A.; Fitch, D. M.; Smith, T. E.; Cee, V. J. Application of Complex Aldol Reactions to the Total Synthesis of Phorboxazole B. J. Am. Chem. Soc. 2000, 122, 10033-10046.<br />
<br />
Gnatt, A. L.; Cramer, P; Fu, J.; Bushnell, D. A.; and Kornberg, R. D. Structural Basis of Transcription: An RNA Polymerase II Elongation Complex at 3.3 Å Resolution. Science. 2001, 292, 1876-1882 1i6h<br />
<br />
Hahn, S. Structure and Mechanism of the RNA Polymerase II Transcription Machinery. Nature Structure and Molecular Biology. 2004, 11, 394-403. <br />
<br />
He, Yuan, et al. Near-atomic resolution visualization of human transcription promoter opening. Nature 533.7603. 2016.<br />
<br />
Nudler, E. RNA Polymerase Active Center: The Molecular Engine of Transcription. Annu. Rev. Biochem. 2009, 78, 335-361.<br />
<br />
Orphanides, George, Thierry Lagrange, and Danny Reinberg. The general transcription factors of RNA polymerase II. Genes & development 10.21. 1996. 2657-2683<br />
<br />
Shah, N. et. al. Tyrosine-1 of RNA Polymerase II CTD Controls Global Termination of Gene Transcription in Mammals. Molecular Cell. 2018, 69, 48-61.<br />
<br />
Uzman, A.; Voet, D. Student companion Fundamentals of biochemistry: life at the molecular level, 4th ed., Donald Voet, Judith G. Voet, Charlotte W. Pratt; John Wiley & amp; Sons, 2012.<br />
<br />
Xu, J.; Lahiri, I.; Wang, W.; Wier, A.; Cianfrocco, M. A.; Chong, J.; Hare, A. A.; Dervan, P. B.; DiMaio, F.; Leschziner, A. E.; Wang, D. Structural Basis for the Initiation of Eukaryotic Transcription-coupled DNA Repair. Nature. 2017. 551, 653-657 5vvr<br />
<br />
Xin, L.; Bushnell, D. A.; and Kornburg, R. D. RNA Polymerase II Transcription: Structure and Mechanism. Biochemica et Biophysica Acta. 2013, 1829, 2-8.<br />
<br />
Yan, C., Dodd, T., He, Y., Tainer, J. A., Tsutakawa, S. E., & Ivanov, I. (2019). Transcription preinitiation complex structure and dynamics provide insight into genetic diseases. Nature Structural and Molecular Biology, 26(6), 397-406.<br />
<br />
Alpha-aminitin chemical structure image courtesy of https://en.wikipedia.org/wiki/Alpha-Amanitin#/media/File:Alpha-amanitin_structure.png<br />
<br />
== Notes ==<br />
From structural components:<br />
<br />
Structural overview: [PDB: 5VVR: with highlighted sections mentioned below]<br />
<br />
Bridge: Depicted: [PDB: 1I6H: 810-845.a]<br />
<br />
Wall: Depicted: [PDB: 1R5U: 853-919.b; 933-972.b]<br />
<br />
Clamp: Depicted: [PDB: 1R5U: 3-345.a; 1395-1435.a; 1158-1124.b]<br />
<br />
Rudder: Depicted: [PDB: 5VVR: 306-321.a]<br />
<br />
<br />
<br />
== Content Donators ==<br />
<br />
This page was created as a final project for the Advanced Biochemistry course at Wabash College during the Fall of 2019. This page was reviewed by Dr. Wally Novak of Wabash College.</div>Wally Novakhttps://proteopedia.org/index.php?title=User:Wally_Novak&diff=3288331User:Wally Novak2020-09-08T12:43:35Z<p>Wally Novak: </p>
<hr />
<div>Associate Professor of Chemistry at Wabash College<br />
*[[User:Wally Novak/Sandbox_Whitaker]]<br />
*[[User:Wally Novak/Sandbox_Miller]]<br />
*[[User:Wally Novak/Sandbox_Hicks]]<br />
*[[User:Wally Novak/Sandbox_Brown]]<br />
*[[User:Wally Novak/Sandbox tutorial]]<br />
*[[User:Wally Novak/Sandbox_RNAPolII_WN]]<br />
*[[User:Wally Novak/Sandbox2_RNAPolII]]</div>Wally Novakhttps://proteopedia.org/index.php?title=RNA_Polymerase_II&diff=3099191RNA Polymerase II2019-10-22T17:15:10Z<p>Wally Novak: </p>
<hr />
<div><StructureSection load='1i6h' size='340' side='right' caption='RNA Polymerase II''><br />
== Introduction ==<br />
RNA polymerase II (RNAP II) is an enzyme that transcribes protein-encoding genes, and it therefore is responsible for the synthesis of mRNA.Brief description of mRNA. Size and composition???<br />
== Structural Components ==<br />
<br />
This section will briefly discuss the chief structural components involved in the mechanism. <br />
<br />
<br />
[[ Image:RnapII struct features.jpg|300px|left|thumb| The clamp (magenta), wall (navy blue), rudder (red), bridge (orange), RNA (light blue), and DNA (blue) are depicted. See below for PDB's and residue numbers.]]<br />
<br />
To begin, the <scene name='82/824648/Clamp/5'>clamp</scene> swings to trap the DNA in the cleft. Further along, the <scene name='82/824648/Wall/2'>wall</scene> sends the DNA template through the cleft in approximately a 90° turn. Both the clamp and wall are parts of the Rpb2 subunit. Further along in the process, the <scene name='82/824648/Rudder/3'>rudder</scene> separates the newly synthesized RNA strand from the DNA template. The DNA reforms into a double helix as it leaves RNA pol II. <br />
<br />
Other components of RNA pol II include the following:<br />
The jaw is the opening through which DNA enters. The funnel is what the NTP’s travel through to be incorporated into the growing RNA strand, and the pore is the end of the funnel. The <scene name='82/824648/Bridge/6'>bridge</scene> is an Rpb1 segment that translocates the DNA-RNA complex at the end of each cycle of catalysis. <scene name='82/824648/Magnesium/3'>Magnesium</scene> is located within the active site and functions as the catalyst. <br />
<br />
== Mechanism of Action ==<br />
<br />
1wcm <scene name='82/824544/Active_site_1/1'>Daniel Andry is a stud</scene><br />
1i6h <scene name='82/824648/Active_site_2/1'>James Daniel Andry is my hero</scene><br />
<br />
== α-Amanitin ==<br />
α-Amanitin is a bicyclic octapeptide that adheres tightly with RNAP II, which blocks the elongation steps. α-amanitin binds in the funnel and interacts with the bridge helix and adjacent Rpb1, but it does not inhibit the RNA pol II’s interaction with NTP. Instead, α-amanitin likely challenges the bridge’s conformational change that is necessary for the purposed RNAP translocation step. α-Amanitin, found in the poisonous mushroom death cap, leads to death after several days. This time frame aligns with the rate at which mRNA’s and proteins turnover. <br />
[[Image:Alpha-amanitin structure (1).png|300px|right|thumb| The chemical structure of α-amanitin.]]<br />
<br />
<br />
== Modifications ==<br />
<br />
== General Transcription Factors ==<br />
<br />
In both eukaryotes and prokaryotes, the basic mechanism for initiating transcription is the same: protein factors selectively bind to promoter regions on DNA. Prokaryotes use sigma factors while eukaryotes use a complex of 6 general transcription factors (GTFs). These GTFs are all named similarly and begin with TF, for transcription factor, followed by the Roman numeral II since they are involved in transcription by RNAP II. The combination of all the transcription factors bound to the DNA promoter region, in complex with RNAP II, is called the preinitiation complex (PIC). The formation of the PIC occurs in an ordered pathway, beginning with the TATA box which is a promoter region on DNA at position -27.<br />
<br />
Process of PIC formation:<br />
<br />
1. <scene name='82/824648/Tfiid-tbp/1'>TFIID</scene> contains a subunit named the TATA-binding protein (TBP), which recognizes and binds to the TATA box on the DNA promoter.<br />
<br />
2. <scene name='82/824648/Tfiib/1'>TFIIB</scene> and <scene name='82/824648/Tfiia/1'>TFIIA</scene> and interact with TBP and are recruted to the promoter.<br />
<br />
3. <scene name='82/824648/Tfiif/1'>TFIIF</scene> binds directly to RNAP II and escorts it to the promoter while <scene name='82/824648/Tfiib/1'>TFIIB</scene> helps the complex bind correctly. <br />
<br />
4. <scene name='82/824648/Tfiie/1'>TFIIE</scene> and <scene name='82/824648/Tfiih/1'>TFIIH</scene> are sequentually recruited which completes the PIC. <br />
<br />
PIC NON-TRANSPARENT COMPLEX<br />
FIGURE CAPTIONS-PDB AND COLOR<br />
CENTERING F<br />
<br />
Once the PIC is formed, RNAP II initiates RNA synthesis and produces a short transcript. When RNAP II becomes phosphorylated, it releases some of the GTFs from the complex and moves away from the promoter. TFIID stays bound to the promoter and can reinitiate transcription. The transcription factors are replaced by a new six-protein complex call the Elongator. TFIIF and TFIIH both remain associated with RNAP II during elongation. <br />
<br />
<br />
<br />
</StructureSection><br />
== References ==<br />
Bushnell, D. A.; Westover, K. D.; Davis, R. E.; Kornberg, R. D. Structural Basis of Transcription: An RNA Polymerase II-TFIIB Cocrystal at 4.5 Angstroms. Science. 2004, 303, 983-988<br />
<br />
Cramer, P.; Bushnell, D. A.; Kornberg, R. D. Structural Basis of Transcription: RNA Polymerase II at 2.8 Ångstrom Resolution. Science. 2001, 292, 1863-1876<br />
<br />
Evans, D. A.; Fitch, D. M.; Smith, T. E.; Cee, V. J. Application of Complex Aldol Reactions to the Total Synthesis of Phorboxazole B. J. Am. Chem. Soc. 2000, 122, 10033-10046.<br />
<br />
Gnatt, A. L.; Cramer, P; Fu, J.; Bushnell, D. A.; and Kornberg, R. D. Structural Basis of Transcription: An RNA Polymerase II Elongation Complex at 3.3 Å Resolution. Science. 2001, 292, 1876-1882 1i6h<br />
<br />
He, Yuan, et al. Near-atomic resolution visualization of human transcription promoter opening. Nature 533.7603. 2016.<br />
<br />
Orphanides, George, Thierry Lagrange, and Danny Reinberg. The general transcription factors of RNA polymerase II. Genes & development 10.21. 1996. 2657-2683<br />
<br />
Uzman, A.; Voet, D. Student companion Fundamentals of biochemistry: life at the molecular level, 4th ed., Donald Voet, Judith G. Voet, Charlotte W. Pratt; John Wiley & amp; Sons, 2012.<br />
<br />
Xu, J.; Lahiri, I.; Wang, W.; Wier, A.; Cianfrocco, M. A.; Chong, J.; Hare, A. A.; Dervan, P. B.; DiMaio, F.; Leschziner, A. E.; Wang, D. Structural Basis for the Initiation of Eukaryotic Transcription-coupled DNA Repair. Nature. 2017. 551, 653-657 5vvr<br />
<br />
Yan, C., Dodd, T., He, Y., Tainer, J. A., Tsutakawa, S. E., & Ivanov, I. (2019). Transcription preinitiation complex structure and dynamics provide insight into genetic diseases. Nature Structural and Molecular Biology, 26(6), 397-406.<br />
<br />
Alpha-aminitin chemical structure image courtesy of https://en.wikipedia.org/wiki/Alpha-Amanitin#/media/File:Alpha-amanitin_structure.png<br />
<br />
== Notes ==<br />
From structural components:<br />
<br />
Structural overview: [PDB: 5VVR: with highlighted sections mentioned below]<br />
<br />
Bridge: Depicted: [PDB: 1I6H: 810-845.a]<br />
<br />
Wall: Depicted: [PDB: 1R5U: 853-919.b; 933-972.b]<br />
<br />
Clamp: Depicted: [PDB: 1R5U: 3-345.a; 1395-1435.a; 1158-1124.b]<br />
<br />
Rudder: Depicted: [PDB: 5VVR: 306-321.a]<br />
<br />
<br />
== Content Donators ==<br />
<br />
This page was created as a final project for the Advanced Biochemistry course at Wabash College during the Fall of 2019. This page was reviewed by Dr. Wally Novak of Wabash College.</div>Wally Novakhttps://proteopedia.org/index.php?title=RNA_Polymerase_II&diff=3099190RNA Polymerase II2019-10-22T17:14:46Z<p>Wally Novak: </p>
<hr />
<div><StructureSection load='1i6h' size='340' side='right' caption='RNA Polymerase II''><br />
<br />
It is weird we have two sections of contents...<br />
<br />
== Introduction ==<br />
RNA polymerase II (RNAP II) is an enzyme that transcribes protein-encoding genes, and it therefore is responsible for the synthesis of mRNA.Brief description of mRNA. Size and composition???<br />
== Structural Components ==<br />
<br />
This section will briefly discuss the chief structural components involved in the mechanism. <br />
<br />
<br />
[[ Image:RnapII struct features.jpg|300px|left|thumb| The clamp (magenta), wall (navy blue), rudder (red), bridge (orange), RNA (light blue), and DNA (blue) are depicted. See below for PDB's and residue numbers.]]<br />
<br />
To begin, the <scene name='82/824648/Clamp/5'>clamp</scene> swings to trap the DNA in the cleft. Further along, the <scene name='82/824648/Wall/2'>wall</scene> sends the DNA template through the cleft in approximately a 90° turn. Both the clamp and wall are parts of the Rpb2 subunit. Further along in the process, the <scene name='82/824648/Rudder/3'>rudder</scene> separates the newly synthesized RNA strand from the DNA template. The DNA reforms into a double helix as it leaves RNA pol II. <br />
<br />
Other components of RNA pol II include the following:<br />
The jaw is the opening through which DNA enters. The funnel is what the NTP’s travel through to be incorporated into the growing RNA strand, and the pore is the end of the funnel. The <scene name='82/824648/Bridge/6'>bridge</scene> is an Rpb1 segment that translocates the DNA-RNA complex at the end of each cycle of catalysis. <scene name='82/824648/Magnesium/3'>Magnesium</scene> is located within the active site and functions as the catalyst. <br />
<br />
== Mechanism of Action ==<br />
<br />
1wcm <scene name='82/824544/Active_site_1/1'>Daniel Andry is a stud</scene><br />
1i6h <scene name='82/824648/Active_site_2/1'>James Daniel Andry is my hero</scene><br />
<br />
== α-Amanitin ==<br />
α-Amanitin is a bicyclic octapeptide that adheres tightly with RNAP II, which blocks the elongation steps. α-amanitin binds in the funnel and interacts with the bridge helix and adjacent Rpb1, but it does not inhibit the RNA pol II’s interaction with NTP. Instead, α-amanitin likely challenges the bridge’s conformational change that is necessary for the purposed RNAP translocation step. α-Amanitin, found in the poisonous mushroom death cap, leads to death after several days. This time frame aligns with the rate at which mRNA’s and proteins turnover. <br />
[[Image:Alpha-amanitin structure (1).png|300px|right|thumb| The chemical structure of α-amanitin.]]<br />
<br />
<br />
== Modifications ==<br />
<br />
== General Transcription Factors ==<br />
<br />
In both eukaryotes and prokaryotes, the basic mechanism for initiating transcription is the same: protein factors selectively bind to promoter regions on DNA. Prokaryotes use sigma factors while eukaryotes use a complex of 6 general transcription factors (GTFs). These GTFs are all named similarly and begin with TF, for transcription factor, followed by the Roman numeral II since they are involved in transcription by RNAP II. The combination of all the transcription factors bound to the DNA promoter region, in complex with RNAP II, is called the preinitiation complex (PIC). The formation of the PIC occurs in an ordered pathway, beginning with the TATA box which is a promoter region on DNA at position -27.<br />
<br />
Process of PIC formation:<br />
<br />
1. <scene name='82/824648/Tfiid-tbp/1'>TFIID</scene> contains a subunit named the TATA-binding protein (TBP), which recognizes and binds to the TATA box on the DNA promoter.<br />
<br />
2. <scene name='82/824648/Tfiib/1'>TFIIB</scene> and <scene name='82/824648/Tfiia/1'>TFIIA</scene> and interact with TBP and are recruted to the promoter.<br />
<br />
3. <scene name='82/824648/Tfiif/1'>TFIIF</scene> binds directly to RNAP II and escorts it to the promoter while <scene name='82/824648/Tfiib/1'>TFIIB</scene> helps the complex bind correctly. <br />
<br />
4. <scene name='82/824648/Tfiie/1'>TFIIE</scene> and <scene name='82/824648/Tfiih/1'>TFIIH</scene> are sequentually recruited which completes the PIC. <br />
<br />
PIC NON-TRANSPARENT COMPLEX<br />
FIGURE CAPTIONS-PDB AND COLOR<br />
CENTERING F<br />
<br />
Once the PIC is formed, RNAP II initiates RNA synthesis and produces a short transcript. When RNAP II becomes phosphorylated, it releases some of the GTFs from the complex and moves away from the promoter. TFIID stays bound to the promoter and can reinitiate transcription. The transcription factors are replaced by a new six-protein complex call the Elongator. TFIIF and TFIIH both remain associated with RNAP II during elongation. <br />
<br />
<br />
<br />
</StructureSection><br />
== References ==<br />
Bushnell, D. A.; Westover, K. D.; Davis, R. E.; Kornberg, R. D. Structural Basis of Transcription: An RNA Polymerase II-TFIIB Cocrystal at 4.5 Angstroms. Science. 2004, 303, 983-988<br />
<br />
Cramer, P.; Bushnell, D. A.; Kornberg, R. D. Structural Basis of Transcription: RNA Polymerase II at 2.8 Ångstrom Resolution. Science. 2001, 292, 1863-1876<br />
<br />
Evans, D. A.; Fitch, D. M.; Smith, T. E.; Cee, V. J. Application of Complex Aldol Reactions to the Total Synthesis of Phorboxazole B. J. Am. Chem. Soc. 2000, 122, 10033-10046.<br />
<br />
Gnatt, A. L.; Cramer, P; Fu, J.; Bushnell, D. A.; and Kornberg, R. D. Structural Basis of Transcription: An RNA Polymerase II Elongation Complex at 3.3 Å Resolution. Science. 2001, 292, 1876-1882 1i6h<br />
<br />
He, Yuan, et al. Near-atomic resolution visualization of human transcription promoter opening. Nature 533.7603. 2016.<br />
<br />
Orphanides, George, Thierry Lagrange, and Danny Reinberg. The general transcription factors of RNA polymerase II. Genes & development 10.21. 1996. 2657-2683<br />
<br />
Uzman, A.; Voet, D. Student companion Fundamentals of biochemistry: life at the molecular level, 4th ed., Donald Voet, Judith G. Voet, Charlotte W. Pratt; John Wiley & amp; Sons, 2012.<br />
<br />
Xu, J.; Lahiri, I.; Wang, W.; Wier, A.; Cianfrocco, M. A.; Chong, J.; Hare, A. A.; Dervan, P. B.; DiMaio, F.; Leschziner, A. E.; Wang, D. Structural Basis for the Initiation of Eukaryotic Transcription-coupled DNA Repair. Nature. 2017. 551, 653-657 5vvr<br />
<br />
Yan, C., Dodd, T., He, Y., Tainer, J. A., Tsutakawa, S. E., & Ivanov, I. (2019). Transcription preinitiation complex structure and dynamics provide insight into genetic diseases. Nature Structural and Molecular Biology, 26(6), 397-406.<br />
<br />
Alpha-aminitin chemical structure image courtesy of https://en.wikipedia.org/wiki/Alpha-Amanitin#/media/File:Alpha-amanitin_structure.png<br />
<br />
== Notes ==<br />
From structural components:<br />
<br />
Structural overview: [PDB: 5VVR: with highlighted sections mentioned below]<br />
<br />
Bridge: Depicted: [PDB: 1I6H: 810-845.a]<br />
<br />
Wall: Depicted: [PDB: 1R5U: 853-919.b; 933-972.b]<br />
<br />
Clamp: Depicted: [PDB: 1R5U: 3-345.a; 1395-1435.a; 1158-1124.b]<br />
<br />
Rudder: Depicted: [PDB: 5VVR: 306-321.a]<br />
<br />
<br />
== Content Donators ==<br />
<br />
This page was created as a final project for the Advanced Biochemistry course at Wabash College during the Fall of 2019. This page was reviewed by Dr. Wally Novak of Wabash College.</div>Wally Novakhttps://proteopedia.org/index.php?title=RNA_Polymerase_II&diff=3099189RNA Polymerase II2019-10-22T17:13:34Z<p>Wally Novak: </p>
<hr />
<div>== RNAP II ==<br />
<StructureSection load='1i6h' size='340' side='right' caption='RNA Polymerase II''><br />
<br />
It is weird we have two sections of contents...<br />
<br />
== Introduction ==<br />
RNA polymerase II (RNAP II) is an enzyme that transcribes protein-encoding genes, and it therefore is responsible for the synthesis of mRNA.Brief description of mRNA. Size and composition???<br />
== Structural Components ==<br />
<br />
This section will briefly discuss the chief structural components involved in the mechanism. <br />
<br />
<br />
[[ Image:RnapII struct features.jpg|300px|left|thumb| The clamp (magenta), wall (navy blue), rudder (red), bridge (orange), RNA (light blue), and DNA (blue) are depicted. See below for PDB's and residue numbers.]]<br />
<br />
To begin, the <scene name='82/824648/Clamp/5'>clamp</scene> swings to trap the DNA in the cleft. Further along, the <scene name='82/824648/Wall/2'>wall</scene> sends the DNA template through the cleft in approximately a 90° turn. Both the clamp and wall are parts of the Rpb2 subunit. Further along in the process, the <scene name='82/824648/Rudder/3'>rudder</scene> separates the newly synthesized RNA strand from the DNA template. The DNA reforms into a double helix as it leaves RNA pol II. <br />
<br />
Other components of RNA pol II include the following:<br />
The jaw is the opening through which DNA enters. The funnel is what the NTP’s travel through to be incorporated into the growing RNA strand, and the pore is the end of the funnel. The <scene name='82/824648/Bridge/6'>bridge</scene> is an Rpb1 segment that translocates the DNA-RNA complex at the end of each cycle of catalysis. <scene name='82/824648/Magnesium/3'>Magnesium</scene> is located within the active site and functions as the catalyst. <br />
<br />
== Mechanism of Action ==<br />
<br />
1wcm <scene name='82/824544/Active_site_1/1'>Daniel Andry is a stud</scene><br />
1i6h <scene name='82/824648/Active_site_2/1'>James Daniel Andry is my hero</scene><br />
<br />
== α-Amanitin ==<br />
α-Amanitin is a bicyclic octapeptide that adheres tightly with RNAP II, which blocks the elongation steps. α-amanitin binds in the funnel and interacts with the bridge helix and adjacent Rpb1, but it does not inhibit the RNA pol II’s interaction with NTP. Instead, α-amanitin likely challenges the bridge’s conformational change that is necessary for the purposed RNAP translocation step. α-Amanitin, found in the poisonous mushroom death cap, leads to death after several days. This time frame aligns with the rate at which mRNA’s and proteins turnover. <br />
[[Image:Alpha-amanitin structure (1).png|300px|right|thumb| The chemical structure of α-amanitin.]]<br />
<br />
<br />
== Modifications ==<br />
<br />
== General Transcription Factors ==<br />
<br />
In both eukaryotes and prokaryotes, the basic mechanism for initiating transcription is the same: protein factors selectively bind to promoter regions on DNA. Prokaryotes use sigma factors while eukaryotes use a complex of 6 general transcription factors (GTFs). These GTFs are all named similarly and begin with TF, for transcription factor, followed by the Roman numeral II since they are involved in transcription by RNAP II. The combination of all the transcription factors bound to the DNA promoter region, in complex with RNAP II, is called the preinitiation complex (PIC). The formation of the PIC occurs in an ordered pathway, beginning with the TATA box which is a promoter region on DNA at position -27.<br />
<br />
Process of PIC formation:<br />
<br />
1. <scene name='82/824648/Tfiid-tbp/1'>TFIID</scene> contains a subunit named the TATA-binding protein (TBP), which recognizes and binds to the TATA box on the DNA promoter.<br />
<br />
2. <scene name='82/824648/Tfiib/1'>TFIIB</scene> and <scene name='82/824648/Tfiia/1'>TFIIA</scene> and interact with TBP and are recruted to the promoter.<br />
<br />
3. <scene name='82/824648/Tfiif/1'>TFIIF</scene> binds directly to RNAP II and escorts it to the promoter while <scene name='82/824648/Tfiib/1'>TFIIB</scene> helps the complex bind correctly. <br />
<br />
4. <scene name='82/824648/Tfiie/1'>TFIIE</scene> and <scene name='82/824648/Tfiih/1'>TFIIH</scene> are sequentually recruited which completes the PIC. <br />
<br />
PIC NON-TRANSPARENT COMPLEX<br />
FIGURE CAPTIONS-PDB AND COLOR<br />
CENTERING F<br />
<br />
Once the PIC is formed, RNAP II initiates RNA synthesis and produces a short transcript. When RNAP II becomes phosphorylated, it releases some of the GTFs from the complex and moves away from the promoter. TFIID stays bound to the promoter and can reinitiate transcription. The transcription factors are replaced by a new six-protein complex call the Elongator. TFIIF and TFIIH both remain associated with RNAP II during elongation. <br />
<br />
<br />
<br />
</StructureSection><br />
== References ==<br />
Bushnell, D. A.; Westover, K. D.; Davis, R. E.; Kornberg, R. D. Structural Basis of Transcription: An RNA Polymerase II-TFIIB Cocrystal at 4.5 Angstroms. Science. 2004, 303, 983-988<br />
<br />
Cramer, P.; Bushnell, D. A.; Kornberg, R. D. Structural Basis of Transcription: RNA Polymerase II at 2.8 Ångstrom Resolution. Science. 2001, 292, 1863-1876<br />
<br />
Evans, D. A.; Fitch, D. M.; Smith, T. E.; Cee, V. J. Application of Complex Aldol Reactions to the Total Synthesis of Phorboxazole B. J. Am. Chem. Soc. 2000, 122, 10033-10046.<br />
<br />
Gnatt, A. L.; Cramer, P; Fu, J.; Bushnell, D. A.; and Kornberg, R. D. Structural Basis of Transcription: An RNA Polymerase II Elongation Complex at 3.3 Å Resolution. Science. 2001, 292, 1876-1882 1i6h<br />
<br />
He, Yuan, et al. Near-atomic resolution visualization of human transcription promoter opening. Nature 533.7603. 2016.<br />
<br />
Orphanides, George, Thierry Lagrange, and Danny Reinberg. The general transcription factors of RNA polymerase II. Genes & development 10.21. 1996. 2657-2683<br />
<br />
Uzman, A.; Voet, D. Student companion Fundamentals of biochemistry: life at the molecular level, 4th ed., Donald Voet, Judith G. Voet, Charlotte W. Pratt; John Wiley & amp; Sons, 2012.<br />
<br />
Xu, J.; Lahiri, I.; Wang, W.; Wier, A.; Cianfrocco, M. A.; Chong, J.; Hare, A. A.; Dervan, P. B.; DiMaio, F.; Leschziner, A. E.; Wang, D. Structural Basis for the Initiation of Eukaryotic Transcription-coupled DNA Repair. Nature. 2017. 551, 653-657 5vvr<br />
<br />
Yan, C., Dodd, T., He, Y., Tainer, J. A., Tsutakawa, S. E., & Ivanov, I. (2019). Transcription preinitiation complex structure and dynamics provide insight into genetic diseases. Nature Structural and Molecular Biology, 26(6), 397-406.<br />
<br />
Alpha-aminitin chemical structure image courtesy of https://en.wikipedia.org/wiki/Alpha-Amanitin#/media/File:Alpha-amanitin_structure.png<br />
<br />
== Notes ==<br />
From structural components:<br />
<br />
Structural overview: [PDB: 5VVR: with highlighted sections mentioned below]<br />
<br />
Bridge: Depicted: [PDB: 1I6H: 810-845.a]<br />
<br />
Wall: Depicted: [PDB: 1R5U: 853-919.b; 933-972.b]<br />
<br />
Clamp: Depicted: [PDB: 1R5U: 3-345.a; 1395-1435.a; 1158-1124.b]<br />
<br />
Rudder: Depicted: [PDB: 5VVR: 306-321.a]<br />
<br />
<br />
== Content Donators ==<br />
<br />
This page was created as a final project for the Advanced Biochemistry course at Wabash College during the Fall of 2019. This page was reviewed by Dr. Wally Novak of Wabash College.</div>Wally Novakhttps://proteopedia.org/index.php?title=RNA_Polymerase_II&diff=3099188RNA Polymerase II2019-10-22T17:11:07Z<p>Wally Novak: </p>
<hr />
<div>== RNAP II ==<br />
<StructureSection load='1i6h' size='340' side='right' caption='RNA Polymerase II''><br />
<br />
== Introduction ==<br />
RNA polymerase II (RNAP II) is an enzyme that transcribes protein-encoding genes, and it therefore is responsible for the synthesis of mRNA.Brief description of mRNA. Size and composition???<br />
== Structural Components ==<br />
<br />
This section will briefly discuss the chief structural components involved in the mechanism. <br />
<br />
<br />
[[ Image:RnapII struct features.jpg|300px|left|thumb| The clamp (magenta), wall (navy blue), rudder (red), bridge (orange), RNA (light blue), and DNA (blue) are depicted. See below for PDB's and residue numbers.]]<br />
<br />
To begin, the <scene name='82/824648/Clamp/5'>clamp</scene> swings to trap the DNA in the cleft. Further along, the <scene name='82/824648/Wall/2'>wall</scene> sends the DNA template through the cleft in approximately a 90° turn. Both the clamp and wall are parts of the Rpb2 subunit. Further along in the process, the <scene name='82/824648/Rudder/3'>rudder</scene> separates the newly synthesized RNA strand from the DNA template. The DNA reforms into a double helix as it leaves RNA pol II. <br />
<br />
Other components of RNA pol II include the following:<br />
The jaw is the opening through which DNA enters. The funnel is what the NTP’s travel through to be incorporated into the growing RNA strand, and the pore is the end of the funnel. The <scene name='82/824648/Bridge/6'>bridge</scene> is an Rpb1 segment that translocates the DNA-RNA complex at the end of each cycle of catalysis. <scene name='82/824648/Magnesium/3'>Magnesium</scene> is located within the active site and functions as the catalyst. <br />
<br />
== Mechanism of Action ==<br />
<br />
1wcm <scene name='82/824544/Active_site_1/1'>Daniel Andry is a stud</scene><br />
1i6h <scene name='82/824648/Active_site_2/1'>James Daniel Andry is my hero</scene><br />
<br />
== α-Amanitin ==<br />
α-Amanitin is a bicyclic octapeptide that adheres tightly with RNAP II, which blocks the elongation steps. α-amanitin binds in the funnel and interacts with the bridge helix and adjacent Rpb1, but it does not inhibit the RNA pol II’s interaction with NTP. Instead, α-amanitin likely challenges the bridge’s conformational change that is necessary for the purposed RNAP translocation step. α-Amanitin, found in the poisonous mushroom death cap, leads to death after several days. This time frame aligns with the rate at which mRNA’s and proteins turnover. <br />
[[Image:Alpha-amanitin structure (1).png|300px|right|thumb| The chemical structure of α-amanitin.]]<br />
<br />
<br />
== Modifications ==<br />
<br />
== General Transcription Factors ==<br />
<br />
In both eukaryotes and prokaryotes, the basic mechanism for initiating transcription is the same: protein factors selectively bind to promoter regions on DNA. Prokaryotes use sigma factors while eukaryotes use a complex of 6 general transcription factors (GTFs). These GTFs are all named similarly and begin with TF, for transcription factor, followed by the Roman numeral II since they are involved in transcription by RNAP II. The combination of all the transcription factors bound to the DNA promoter region, in complex with RNAP II, is called the preinitiation complex (PIC). The formation of the PIC occurs in an ordered pathway, beginning with the TATA box which is a promoter region on DNA at position -27.<br />
<br />
Process of PIC formation:<br />
<br />
1. <scene name='82/824648/Tfiid-tbp/1'>TFIID</scene> contains a subunit named the TATA-binding protein (TBP), which recognizes and binds to the TATA box on the DNA promoter.<br />
<br />
2. <scene name='82/824648/Tfiib/1'>TFIIB</scene> and <scene name='82/824648/Tfiia/1'>TFIIA</scene> and interact with TBP and are recruted to the promoter.<br />
<br />
3. <scene name='82/824648/Tfiif/1'>TFIIF</scene> binds directly to RNAP II and escorts it to the promoter while <scene name='82/824648/Tfiib/1'>TFIIB</scene> helps the complex bind correctly. <br />
<br />
4. <scene name='82/824648/Tfiie/1'>TFIIE</scene> and <scene name='82/824648/Tfiih/1'>TFIIH</scene> are sequentually recruited which completes the PIC. <br />
<br />
PIC NON-TRANSPARENT COMPLEX<br />
FIGURE CAPTIONS-PDB AND COLOR<br />
CENTERING F<br />
<br />
Once the PIC is formed, RNAP II initiates RNA synthesis and produces a short transcript. When RNAP II becomes phosphorylated, it releases some of the GTFs from the complex and moves away from the promoter. TFIID stays bound to the promoter and can reinitiate transcription. The transcription factors are replaced by a new six-protein complex call the Elongator. TFIIF and TFIIH both remain associated with RNAP II during elongation. <br />
<br />
<br />
<br />
</StructureSection><br />
== References ==<br />
Bushnell, D. A.; Westover, K. D.; Davis, R. E.; Kornberg, R. D. Structural Basis of Transcription: An RNA Polymerase II-TFIIB Cocrystal at 4.5 Angstroms. Science. 2004, 303, 983-988<br />
<br />
Cramer, P.; Bushnell, D. A.; Kornberg, R. D. Structural Basis of Transcription: RNA Polymerase II at 2.8 Ångstrom Resolution. Science. 2001, 292, 1863-1876<br />
<br />
Evans, D. A.; Fitch, D. M.; Smith, T. E.; Cee, V. J. Application of Complex Aldol Reactions to the Total Synthesis of Phorboxazole B. J. Am. Chem. Soc. 2000, 122, 10033-10046.<br />
<br />
Gnatt, A. L.; Cramer, P; Fu, J.; Bushnell, D. A.; and Kornberg, R. D. Structural Basis of Transcription: An RNA Polymerase II Elongation Complex at 3.3 Å Resolution. Science. 2001, 292, 1876-1882 1i6h<br />
<br />
He, Yuan, et al. Near-atomic resolution visualization of human transcription promoter opening. Nature 533.7603. 2016.<br />
<br />
Orphanides, George, Thierry Lagrange, and Danny Reinberg. The general transcription factors of RNA polymerase II. Genes & development 10.21. 1996. 2657-2683<br />
<br />
Uzman, A.; Voet, D. Student companion Fundamentals of biochemistry: life at the molecular level, 4th ed., Donald Voet, Judith G. Voet, Charlotte W. Pratt; John Wiley & amp; Sons, 2012.<br />
<br />
Xu, J.; Lahiri, I.; Wang, W.; Wier, A.; Cianfrocco, M. A.; Chong, J.; Hare, A. A.; Dervan, P. B.; DiMaio, F.; Leschziner, A. E.; Wang, D. Structural Basis for the Initiation of Eukaryotic Transcription-coupled DNA Repair. Nature. 2017. 551, 653-657 5vvr<br />
<br />
Yan, C., Dodd, T., He, Y., Tainer, J. A., Tsutakawa, S. E., & Ivanov, I. (2019). Transcription preinitiation complex structure and dynamics provide insight into genetic diseases. Nature Structural and Molecular Biology, 26(6), 397-406.<br />
<br />
Alpha-aminitin chemical structure image courtesy of https://en.wikipedia.org/wiki/Alpha-Amanitin#/media/File:Alpha-amanitin_structure.png<br />
<br />
== Notes ==<br />
From structural components:<br />
<br />
Structural overview: [PDB: 5VVR: with highlighted sections mentioned below]<br />
<br />
Bridge: Depicted: [PDB: 1I6H: 810-845.a]<br />
<br />
Wall: Depicted: [PDB: 1R5U: 853-919.b; 933-972.b]<br />
<br />
Clamp: Depicted: [PDB: 1R5U: 3-345.a; 1395-1435.a; 1158-1124.b]<br />
<br />
Rudder: Depicted: [PDB: 5VVR: 306-321.a]<br />
<br />
<br />
== Content Donators ==<br />
<br />
This page was created as a final project for the Advanced Biochemistry course at Wabash College during the Fall of 2019. This page was reviewed by Dr. Wally Novak of Wabash College.</div>Wally Novakhttps://proteopedia.org/index.php?title=RNA_Polymerase_II&diff=3093046RNA Polymerase II2019-10-08T13:11:35Z<p>Wally Novak: </p>
<hr />
<div>== RNAP II ==<br />
<StructureSection load='1i6h' size='340' side='right' caption='RNA Polymerase II''><br />
<br />
== Introduction ==<br />
Titus wrote this<br />
<br />
The cookie monster is now the veggie monster. <br />
<br />
== Structural Components ==<br />
<br />
Like in much of biochemistry, the structure of RNAP II gives insights into its function. <br />
<br />
This is an image with all of the following discussed structures within one image.<br />
<br />
<br />
The <scene name='82/824648/Clamp/2'>clamp</scene> swings to trap the DNA in the cleft. Further along, the <scene name='82/824648/Wall/1'>wall</scene> sends the DNA template through the cleft in approximately a 90° turn. Both the clamp and wall are parts of the Rpb2 subunit. Further along in the process, the <scene name='82/824648/Rudder/1'>rudder</scene> separates the newly synthesized RNA strand from the DNA template. The DNA reforms into a double helix as it leaves RNA pol II. <br />
<br />
Other components of RNA pol II include the following:<br />
The jaw is the opening through which DNA enters. The funnel is what the NTP’s travel through to be incorporated into the growing RNA strand, and the pore is the end of the funnel. The <scene name='82/824648/Bridge/4'>bridge</scene> is an Rpb1 segment that translocates the DNA-RNA complex at the end of each cycle of catalysis. <scene name='82/824648/Magnesium/1'>Magnesium</scene> is located within the active site and functions as the catalyst. <br />
<br />
== Mechanism of Action ==<br />
<br />
1wcm <scene name='82/824544/Active_site_1/1'>TextToBeDisplayed</scene><br />
1i6h <scene name='82/824648/Active_site_2/1'>TextToBeDisplayed</scene><br />
<br />
== Alpha Amanitin ==<br />
α-Amanitin is a bicyclic octapeptide that adheres tightly with RNAP II, which blocks the elongation steps. α-amanitin binds in the funnel and interacts with the bridge helix and adjacent Rpb1, but it does not inhibit the RNA pol II’s interaction with NTP. Instead, α-amanitin likely challenges the bridge’s conformational change that is necessary for the purposed RNAP translocation step. α-Amanitin, found in the poisonous mushroom death cap, leads to death after several days. This time frame aligns with the rate at which mRNA’s and proteins turnover.<br />
<br />
== Modifications ==<br />
<br />
== General Transcription Factors ==<br />
<br />
In both eukaryotes and prokaryotes, the basic mechanism for initiating transcription is the same: protein factors selectively bind to promoter regions on DNA. Prokaryotes use sigma factors while eukaryotes use a complex of 6 general transcription factors (GTFs). These GTFs are all named similarly and begin with TF, for transcription factor, followed by the Roman numeral II since they are involved in transcription by RNAP II. The combination of all the transcription factors bound to the DNA promoter region, in complex with RNAP II, is called the preinitiation complex (PIC). The formation of the PIC occurs in an ordered pathway, beginning with the TATA box which is a promoter region on DNA at position -27.<br />
<br />
Process of PIC formation:<br />
<br />
1. <scene name='82/824648/Tfiid-tbp/1'>TFIID</scene> contains a subunit named the TATA-binding protein (TBP), which recognizes and binds to the TATA box on the DNA promoter.<br />
<br />
2. <scene name='82/824648/Tfiia/1'>TFIIA</scene> and <scene name='82/824648/Tfiib/1'>TFIIB</scene> interact with TBP and are recruted to the promoter.<br />
<br />
3. <scene name='82/824648/Tfiif/1'>TFIIF</scene> binds directly to RNAP II and escorts it to the promoter while TFIIB helps the complex bind correctly. <br />
<br />
4. <scene name='82/824648/Tfiie/1'>TFIIE</scene> and <scene name='82/824648/Tfiih/1'>TFIIH</scene> are sequentually recruited which completes the PIC. <br />
<br />
Once the PIC is formed, RNAP II initiates RNA synthesis and produces a short transcript. When RNAP II becomes phosphorylated, it releases some of the GTFs from the complex and moves away from the promoter. TFIID stays bound to the promoter and can reinitiate transcription. The transcription factors are replaced by a new six-protein complex call the Elongator. TFIIF and TFIIH both remain associated with RNAP II during elongation. <br />
<br />
<br />
<br />
</StructureSection><br />
== References ==<br />
Bushnell, D. A.; Westover, K. D.; Davis, R. E.; Kornberg, R. D. Structural Basis of Transcription: An RNA Polymerase II-TFIIB Cocrystal at 4.5 Angstroms. Science. 2004, 303, 983-988<br />
<br />
Cramer, P.; Bushnell, D. A.; Kornberg, R. D. Structural Basis of Transcription: RNA Polymerase II at 2.8 Ångstrom Resolution. Science. 2001, 292, 1863-1876<br />
<br />
Evans, D. A.; Fitch, D. M.; Smith, T. E.; Cee, V. J. Application of Complex Aldol Reactions to the Total Synthesis of Phorboxazole B. J. Am. Chem. Soc. 2000, 122, 10033-10046.<br />
<br />
He, Yuan, et al. Near-atomic resolution visualization of human transcription promoter opening. Nature 533.7603. 2016.<br />
<br />
Uzman, A.; Voet, D. Student companion Fundamentals of biochemistry: life at the molecular level, 4th ed., Donald Voet, Judith G. Voet, Charlotte W. Pratt; John Wiley & amp; Sons, 2012.<br />
<br />
Yan, C., Dodd, T., He, Y., Tainer, J. A., Tsutakawa, S. E., & Ivanov, I. (2019). Transcription preinitiation complex structure and dynamics provide insight into genetic diseases. Nature Structural and Molecular Biology, 26(6), 397-406.<br />
<br />
<br />
== Notes ==<br />
From structural components:<br />
<br />
Structural overview: [PDB: 5VVR: with highlighted sections mentioned below]<br />
<br />
Bridge: Depicted: [PDB: 1I6H: 810-845.a]<br />
<br />
Wall: Depicted: [PDB: 1R5U: 853-919.b; 933-972.b]<br />
<br />
Clamp: Depicted: [PDB: 1R5U: 3-345.a; 1395-1435.a; 1158-1124.b]<br />
<br />
Rudder: Depicted: [PDB: 5VVR: 306-321.a]<br />
<br />
<br />
== Content Donators ==<br />
<br />
This page was created as a final project for the Advanced Biochemistry course at Wabash College during the Fall of 2019. This page was reviewed by Dr. Wally Novak of Wabash College.</div>Wally Novakhttps://proteopedia.org/index.php?title=RNA_Polymerase_II&diff=3093041RNA Polymerase II2019-10-08T12:15:48Z<p>Wally Novak: </p>
<hr />
<div>== RNAP II ==<br />
<StructureSection load='1i6h' size='340' side='right' caption='RNA Polymerase II''><br />
<br />
== Introduction ==<br />
<br />
== Structural Components ==<br />
The <scene name='82/824648/Clamp/2'>clamp</scene> swings to trap the DNA in the cleft. Further along, the <scene name='82/824648/Wall/1'>wall</scene> sends the DNA template through the cleft in approximately a 90° turn. Both the clamp and wall are parts of the Rpb2 subunit. Further along in the process, the <scene name='82/824648/Rudder/1'>rudder</scene> separates the newly synthesized RNA strand from the DNA template. The DNA reforms into a double helix as it leaves RNA pol II. <br />
<br />
Other components of RNA pol II include the following:<br />
The jaw is the opening through which DNA enters. The funnel is what the NTP’s travel through to be incorporated into the growing RNA strand, and the pore is the end of the funnel. The <scene name='82/824648/Bridge/4'>bridge</scene> is an Rpb1 segment that translocates the DNA-RNA combination at the end of each cycle of catalysis. <scene name='82/824648/Magnesium/1'>Magnesium</scene> is located within the active site and functions as the catalyst. <br />
<br />
== Mechanism of Action ==<br />
<br />
1wcm <scene name='82/824544/Active_site_1/1'>TextToBeDisplayed</scene><br />
1i6h <scene name='82/824648/Active_site_2/1'>TextToBeDisplayed</scene><br />
<br />
== Alpha Amanitin ==<br />
α-Amanitin is a bicyclic octapeptide that adheres tightly with RNAP II, which blocks the elongation steps. α-amanitin binds in the funnel and interacts with the bridge helix and adjacent Rpb1, but it does not inhibit the RNA pol II’s interaction with NTP. Instead, α-amanitin likely challenges the bridge’s conformational change that is necessary for the purposed RNAP translocation step. α-Amanitin, found in the poisonous mushroom death cap, leads to death after several days. This time frame aligns with the rate at which mRNA’s and proteins turnover.<br />
<br />
== Modifications ==<br />
<br />
== General Transcription Factors ==<br />
<br />
In both eukaryotes and prokaryotes, the basic mechanism for initiating transcription is the same: protein factors selectively bind to promoter regions on DNA. Prokaryotes use sigma factors while eukaryotes use a complex of 6 general transcription factors (GTFs). These GTFs are all named similarly and begin with TF, for transcription factor, followed by the Roman numeral II since they are involved in transcription by RNAP II. The combination of all the transcription factors bound to the DNA promoter region, in complex with RNAP II, is called the preinitiation complex (PIC). The formation of the PIC occurs in an ordered pathway, beginning with the TATA box which is a promoter region on DNA at position -27.<br />
<br />
Process of PIC formation:<br />
<br />
1. <scene name='82/824648/Tfiid-tbp/1'>TFIID</scene> contains a subunit named the TATA-binding protein (TBP), which recognizes and binds to the TATA box on the DNA promoter.<br />
<br />
2. <scene name='82/824648/Tfiia/1'>TFIIA</scene> and <scene name='82/824648/Tfiib/1'>TFIIB</scene> interact with TBP and are recruted to the promoter.<br />
<br />
3. <scene name='82/824648/Tfiif/1'>TFIIF</scene> binds directly to RNAP II and escorts it to the promoter while TFIIB helps the complex bind correctly. <br />
<br />
4. <scene name='82/824648/Tfiie/1'>TFIIE</scene> and <scene name='82/824648/Tfiih/1'>TFIIH</scene> are sequentually recruited which completes the PIC. <br />
<br />
Once the PIC is formed, RNAP II initiates RNA synthesis and produces a short transcript. When RNAP II becomes phosphorylated, it releases some of the GTFs from the complex and moves away from the promoter. TFIID stays bound to the promoter and can reinitiate transcription. The transcription factors are replaced by a new six-protein complex call the Elongator. TFIIF and TFIIH both remain associated with RNAP II during elongation. <br />
<br />
<br />
<br />
</StructureSection><br />
== References ==<br />
Bushnell, D. A.; Westover, K. D.; Davis, R. E.; Kornberg, R. D. Structural Basis of Transcription: An RNA Polymerase II-TFIIB Cocrystal at 4.5 Angstroms. Science. 2004, 303, 983-988<br />
<br />
Cramer, P.; Bushnell, D. A.; Kornberg, R. D. Structural Basis of Transcription: RNA Polymerase II at 2.8 Ångstrom Resolution. Science. 2001, 292, 1863-1876<br />
<br />
Uzman, A.; Voet, D. Student companion Fundamentals of biochemistry: life at the molecular level, 4th ed., Donald Voet, Judith G. Voet, Charlotte W. Pratt; John Wiley & amp; Sons, 2012.<br />
<br />
Evans, D. A.; Fitch, D. M.; Smith, T. E.; Cee, V. J. Application of Complex Aldol Reactions to the Total Synthesis of Phorboxazole B. J. Am. Chem. Soc. 2000, 122, 10033-10046.<br />
<br />
Yan, C., Dodd, T., He, Y., Tainer, J. A., Tsutakawa, S. E., & Ivanov, I. (2019). Transcription preinitiation complex structure and dynamics provide insight into genetic diseases. Nature Structural and Molecular Biology, 26(6), 397-406.<br />
== Notes ==<br />
From structural components:<br />
Bridge: Depicted: [PDB: 1I6H: 810-845.a]<br />
Wall: Depicted: [PDB: 1R5U: 853-919.b; 933-972.b]<br />
Clamp: Depicted: [PDB: 1R5U: 3-345.a; 1395-1435.a; 1158-1124.b]<br />
Rudder: Depicted: [PDB: 5VVR: 306-321.a]<br />
<br />
<br />
== Content Donators ==<br />
<br />
This page was created as a final project for the Advanced Biochemistry course at Wabash College during the Fall of 2019. This page was reviewed by Dr. Wally Novak of Wabash College.</div>Wally Novakhttps://proteopedia.org/index.php?title=User:Wally_Novak/Sandbox_RNAPolII_WN&diff=3088229User:Wally Novak/Sandbox RNAPolII WN2019-09-12T13:26:47Z<p>Wally Novak: </p>
<hr />
<div>==Your Heading Here (maybe something like 'Structure')==<br />
<StructureSection load='2o5i' size='340' side='right' caption='RNAPol II elongation complex' scene=''><br />
This is a default text for your page '''Wally Novak/Sandbox RNAPolII WN'''. Click above on '''edit this page''' to modify. Be careful with the &lt; and &gt; signs.<br />
You may include any references to papers as in: the use of JSmol in Proteopedia <ref>DOI 10.1002/ijch.201300024</ref> or to the article describing Jmol <ref>PMID:21638687</ref> to the rescue.<br />
<br />
== Function ==<br />
<scene name='82/824542/Tacrine_closeup/1'>this zooms in on tacrine</scene><br />
catalytic histidine<br />
== Disease ==<br />
<br />
== Relevance ==<br />
<br />
== Structural highlights ==<br />
<br />
This is a sample scene created with SAT to <scene name="/12/3456/Sample/1">color</scene> by Group, and another to make <scene name="/12/3456/Sample/2">a transparent representation</scene> of the protein. You can make your own scenes on SAT starting from scratch or loading and editing one of these sample scenes.<br />
<br />
</StructureSection><br />
== References ==<br />
<references/></div>Wally Novakhttps://proteopedia.org/index.php?title=File:2o5i_edit.pdb&diff=3088219File:2o5i edit.pdb2019-09-12T13:14:50Z<p>Wally Novak: </p>
<hr />
<div></div>Wally Novakhttps://proteopedia.org/index.php?title=User:Wally_Novak/Sandbox_RNAPolII_WN&diff=3088213User:Wally Novak/Sandbox RNAPolII WN2019-09-12T12:56:28Z<p>Wally Novak: </p>
<hr />
<div>==Your Heading Here (maybe something like 'Structure')==<br />
<StructureSection load='2o5i' size='340' side='right' caption='RNAPol II elongation complex' scene=''><br />
This is a default text for your page '''Wally Novak/Sandbox RNAPolII WN'''. Click above on '''edit this page''' to modify. Be careful with the &lt; and &gt; signs.<br />
You may include any references to papers as in: the use of JSmol in Proteopedia <ref>DOI 10.1002/ijch.201300024</ref> or to the article describing Jmol <ref>PMID:21638687</ref> to the rescue.<br />
<br />
== Function ==<br />
<scene name='82/824542/Tacrine_closeup/1'>this zooms in on tacrine</scene><br />
== Disease ==<br />
<br />
== Relevance ==<br />
<br />
== Structural highlights ==<br />
<br />
This is a sample scene created with SAT to <scene name="/12/3456/Sample/1">color</scene> by Group, and another to make <scene name="/12/3456/Sample/2">a transparent representation</scene> of the protein. You can make your own scenes on SAT starting from scratch or loading and editing one of these sample scenes.<br />
<br />
</StructureSection><br />
== References ==<br />
<references/></div>Wally Novakhttps://proteopedia.org/index.php?title=User:Wally_Novak/Sandbox_RNAPolII_WN&diff=3088207User:Wally Novak/Sandbox RNAPolII WN2019-09-12T12:35:42Z<p>Wally Novak: New page: ==Your Heading Here (maybe something like 'Structure')== <StructureSection load='2o5i' size='340' side='right' caption='RNAPol II elongation complex' scene=''> This is a default text for y...</p>
<hr />
<div>==Your Heading Here (maybe something like 'Structure')==<br />
<StructureSection load='2o5i' size='340' side='right' caption='RNAPol II elongation complex' scene=''><br />
This is a default text for your page '''Wally Novak/Sandbox RNAPolII WN'''. Click above on '''edit this page''' to modify. Be careful with the &lt; and &gt; signs.<br />
You may include any references to papers as in: the use of JSmol in Proteopedia <ref>DOI 10.1002/ijch.201300024</ref> or to the article describing Jmol <ref>PMID:21638687</ref> to the rescue.<br />
<br />
== Function ==<br />
<br />
== Disease ==<br />
<br />
== Relevance ==<br />
<br />
== Structural highlights ==<br />
<br />
This is a sample scene created with SAT to <scene name="/12/3456/Sample/1">color</scene> by Group, and another to make <scene name="/12/3456/Sample/2">a transparent representation</scene> of the protein. You can make your own scenes on SAT starting from scratch or loading and editing one of these sample scenes.<br />
<br />
</StructureSection><br />
== References ==<br />
<references/></div>Wally Novakhttps://proteopedia.org/index.php?title=User:Wally_Novak&diff=3088202User:Wally Novak2019-09-12T12:31:59Z<p>Wally Novak: </p>
<hr />
<div>Associate Professor of Chemistry at Wabash College<br />
*[[User:Wally Novak/Sandbox_Whitaker]]<br />
*[[User:Wally Novak/Sandbox_Miller]]<br />
*[[User:Wally Novak/Sandbox_Hicks]]<br />
*[[User:Wally Novak/Sandbox_Brown]]<br />
*[[User:Wally Novak/Sandbox tutorial]]<br />
*[[User:Wally Novak/Sandbox_RNAPolII_WN]]</div>Wally Novakhttps://proteopedia.org/index.php?title=Sandbox_RDE-1&diff=2955066Sandbox RDE-12018-10-09T04:41:48Z<p>Wally Novak: </p>
<hr />
<div>== Introduction ==<br />
<Structure load='4krf' size='350' frame='true' align='right' caption='Structure of Human Argonaute-1 let-7 complex as representation of RDE-1 since they have similar function. Thus, this complex is a model for Argonaute proteins in general.(PMID: 23746446)' scene='Insert optional scene name here' /><br />
The rde-1 gene is a member of the ''Argonaute'' gene family. Proteins from "[[Argonaute]]" family form an evolutionarily conserved family whose members silence gene expression in pathways such as RNA interference (RNAi). Argonaute family proteins can be divided into two types, AGO and Piwi proteins, depending on the small RNA they bonded to. Both types of Argonaute proteins bind 21–35 nucleotide-long small RNA guides whose sequence identifies the genes to be silenced.<ref name=four/><br />
'''RDE-1''' (RNAi-DEfective 1), a primary Argonaute protein, is required for RNA-mediated interference in ''Caenorhabditis elegans''; thus, it is also known as RNAi promoting factor. Its gene locus was first characterized in ''C. elegans'' mutants resistant to RNAi, and was found to be a member of the Piwi gene family that includes plant, Drosophila, and vertebrate homologs.<ref name=two>PMID: 10535731</ref><br />
<br />
== Structure ==<br />
All "[[Argonaute]]"/Piwi proteins possess three primary/main domains forming a crescent-shaped base: the PAZ, MID, and <scene name='79/798387/Piwi_domain/1'>PIWI</scene> domains. Other structural feature includes the N domain. The amino-terminal PAZ domain uses its oligonucleotide-binding (OB) fold to secure the 3′ end of the small RNA guide strand to ''Argonaute'' protein. A conserved hydrophobic cavity within the PAZ domain recognizes the characteristic two-nucleotide, 3′ overhanging end of the guide-passenger siRNA generated by Dicer. The MID domain anchors the 5′ monophosphate of a siRNA to the ''Argonaute'' protein, securing the guide through multiple cycles of target cleavage. In vitro, studies suggest that 5′ phosphate binding helps align the small RNA on the surface of "Argonaute" protein, ensuring that the correct bond of the target is positioned in the endonuclease active site.<ref name=four>PMID: 21683893</ref> In other words, the PAZ and MID domains orient and anchor the double-stranded siRNA by binding to the 3’ and 5’ termini, respectively, leaving the internal nucleotides accessible for base pairing.<ref name=four/><ref name=three>PMID:15284453</ref> The carboxy-terminal PIWI domain resembles nuclease RNase H in which it folds into an RNase H-like structure. In RDE-1, this domain contains three conserved amino acids, aspartate-aspartate-histidine, that form a catalytic triad "DDH".<ref name=three/> The crystal structure of RDE-1 has not been fully elucidated, but can be assumed to closely resemble its human homologs. The full length of RDE-1 protein is 1020 amino acids (aa)<ref name=two/> in which about 110 of those aa makes up the PAZ domain and 300 aa makes up the PIWI domain.<ref> DOI: 10.1016/s0968-0004(00)01641-8</ref><br />
<br />
== Function ==<br />
[[Image:RDE-1 Proposed Fxn.png|300px||left|thumb|Proposed RNAi pathway for exogenous trigger dsRNA in C. elegans.<ref>https://en.wikipedia.org/wiki/RDE-1#/media/File:Exogenous_RNAi_Pathway_in_C._elegans,_edited.svg</ref>]] RDE-1 is not required for the initial processing of the trigger RNA into siRNAs in RNAi, but it is required in the effector step.<ref>PMID: 11680844</ref> The trigger dsRNA is bound by RDE-4 onto a [[Dicer]] complex and this complex cleaves the dsRNA into 21-25nt primary siRNA. The siRNA binds to RDE-1 promoting the formation of the RNA-induced silencing complex (RISC) and RDE-1 shuttles the siRNA to that effector complex.<ref name=promo>DOI: 10.1016/S0092-8674(02)00793-6</ref><ref name=shuttle>PMID: 12110901</ref> The RNase H activity in PIWI domain in RDE-1 facilitates siRNA maturation, cleaving siRNA into a single stranded siRNA or guide RNA<ref name=cleave>DOI: 10.1038/nsmb.1541</ref> while RISC is activated when ATP is added to the complex and utilized the guide RNA on RDE-1 to base pair with the target transcript.<ref name=shuttle/> The activation of RISC promotes the recruitment of RNA-dependent RNA polymerase (RdRP) which triggers the amplification of the secondary siRNAs to exhibit target transcript degradation.<ref>DOI: 10.1007/978-3-540-75157-1_2</ref><br />
<br />
== Importance ==<br />
Mutation of any residues in the RNase H catalytic triad abolishes Slicer activity in human Argonaute protein Ago2, suggesting that the RNase H domain is directly responsible for target mRNA degradation.<ref> PMID: 15284456</ref> However, RDE-1 has not been implicated in mRNA-cleavage activity.<br />
<br />
Instead, RDE-1 with mutations in the conserved DDH motif exhibit reduced passenger (sense) strand turnover, suggesting that RNase H activity serves to cleave the passenger strand, leaving the guide (antisense) strand accessible for base-pairing to target mRNA.<ref name=cleave/> Further, target silencing can be fully restored in DDH motif mutants by loading single-stranded siRNA, suggesting that a downstream component in the RNAi pathway is responsible for Slicer activity.<ref name=cleave/> Thus, RDE-1’s RNase H domain facilitates siRNA maturation but is not directly involved in cleaving target mRNA transcripts.<br />
<br />
RDE-1 is important for cleavage of the passenger strand and shuttling the siRNA to the appropriate RISC in ''C. elegans''.<ref name=cleave/><ref name=shuttle/> Without RDE-1, RISC cannot obtain the guide RNA for identifying the target transcript and thus transcript degradation cannot process properly or not at all. This protein is therefore also important for silencing a disease-causing gene by degradation of mRNA.<ref name=shuttle/><br />
<br />
<br />
<br />
<br />
== References ==<br />
<references/></div>Wally Novakhttps://proteopedia.org/index.php?title=Sandbox_RDE-1&diff=2955065Sandbox RDE-12018-10-09T04:25:13Z<p>Wally Novak: </p>
<hr />
<div>== Introduction ==<br />
<Structure load='4krf' size='350' frame='true' align='right' caption='Structure of Human Argonaute-1 let-7 complex as representation of RDE-1 since they have similar function. Thus, this complex is a model for Argonaute proteins in general.(PMID: 23746446)' scene='Insert optional scene name here' /><br />
The rde-1 gene is a member of the ''Argonaute'' gene family. Proteins from "Argonaute" family form an evolutionarily conserved family whose members silence gene expression in pathways such as RNA interference (RNAi). Argonaute family proteins can be divided into two types, AGO and Piwi proteins, depending on the small RNA they bonded to. Both types of Argonaute proteins bind 21–35 nucleotide-long small RNA guides whose sequence identifies the genes to be silenced.<ref name=four/><br />
'''RDE-1''' (RNAi-DEfective 1), a primary Argonaute protein, is required for RNA-mediated interference in ''Caenorhabditis elegans''; thus, it is also known as RNAi promoting factor. Its gene locus was first characterized in ''C. elegans'' mutants resistant to RNAi, and was found to be a member of the Piwi gene family that includes plant, Drosophila, and vertebrate homologs.<ref name=two>PMID: 10535731</ref><br />
<br />
== Structure ==<br />
All "Argonaute"/Piwi proteins possess three primary domains forming a crescent-shaped base: the PAZ, MID, and <scene name='79/798387/Piwi_domain/1'>PIWI</scene> domains. The amino-terminal PAZ domain uses its oligonucleotide-binding (OB) fold to secure the 3′ end of the small RNA guide strand to ''Argonaute'' protein. A conserved hydrophobic cavity within the PAZ domain recognizes the characteristic two-nucleotide, 3′ overhanging end of the guide-passenger siRNA generated by Dicer. The MID domain anchors the 5′ monophosphate of a siRNA to the ''Argonaute'' protein, securing the guide through multiple cycles of target cleavage. In vitro, studies suggest that 5′ phosphate binding helps align the small RNA on the surface of "Argonaute" protein, ensuring that the correct bond of the target is positioned in the endonuclease active site.<ref name=four>PMID: 21683893</ref> In other words, the PAZ and MID domains orient and anchor the double-stranded siRNA by binding to the 3’ and 5’ termini, respectively, leaving the internal nucleotides accessible for base pairing.<ref name=four/><ref name=three>PMID:15284453</ref> The carboxy-terminal PIWI domain resembles nuclease RNase H in which it folds into an RNase H-like structure. In RDE-1, this domain contains three conserved amino acids, aspartate-aspartate-histidine, that form a catalytic triad "DDH".<ref name=three/> The crystal structure of RDE-1 has not been fully elucidated, but can be assumed to closely resemble its human homologs. The full length of RDE-1 protein is 1020 amino acids (aa)<ref name=two/> in which about 110 of those aa makes up the PAZ domain and 300 aa makes up the PIWI domain.<ref> DOI: 10.1016/s0968-0004(00)01641-8</ref><br />
<br />
== Function ==<br />
[[Image:RDE-1 Proposed Fxn.png|300px||left|thumb|Proposed RNAi pathway for exogenous trigger dsRNA in C. elegans.<ref>https://en.wikipedia.org/wiki/RDE-1#/media/File:Exogenous_RNAi_Pathway_in_C._elegans,_edited.svg</ref>]] RDE-1 is not required for the initial processing of the trigger RNA into siRNAs in RNAi, but it is required in the effector step.<ref>PMID: 11680844</ref> The trigger dsRNA is bound by RDE-4 onto a [[Dicer]] complex and this complex cleaves the dsRNA into 21-25nt primary siRNA. The siRNA binds to RDE-1 promoting the formation of the RNA-induced silencing complex (RISC) and RDE-1 shuttles the siRNA to that effector complex.<ref name=promo>DOI: 10.1016/S0092-8674(02)00793-6</ref><ref name=shuttle>PMID: 12110901</ref> The RNase H activity in PIWI domain in RDE-1 facilitates siRNA maturation, cleaving siRNA into a single stranded siRNA or guide RNA<ref name=cleave>DOI: 10.1038/nsmb.1541</ref> while RISC is activated when ATP is added to the complex and utilized the guide RNA on RDE-1 to base pair with the target transcript.<ref name=shuttle/> The activation of RISC promotes the recruitment of RNA-dependent RNA polymerase (RdRP) which triggers the amplification of the secondary siRNAs to exhibit target transcript degradation.<ref>DOI: 10.1007/978-3-540-75157-1_2</ref><br />
<br />
== Importance ==<br />
Mutation of any residues in the RNase H catalytic triad abolishes Slicer activity in human Argonaute protein Ago2, suggesting that the RNase H domain is directly responsible for target mRNA degradation.<ref> PMID: 15284456</ref> However, RDE-1 has not been implicated in mRNA-cleavage activity.<br />
<br />
Instead, RDE-1 with mutations in the conserved DDH motif exhibit reduced passenger (sense) strand turnover, suggesting that RNase H activity serves to cleave the passenger strand, leaving the guide (antisense) strand accessible for base-pairing to target mRNA.<ref name=cleave/> Further, target silencing can be fully restored in DDH motif mutants by loading single-stranded siRNA, suggesting that a downstream component in the RNAi pathway is responsible for Slicer activity.<ref name=cleave/> Thus, RDE-1’s RNase H domain facilitates siRNA maturation but is not directly involved in cleaving target mRNA transcripts.<br />
<br />
RDE-1 is important for cleavage of the passenger strand and shuttling the siRNA to the appropriate RISC in ''C. elegans''.<ref name=cleave/><ref name=shuttle/> Without RDE-1, RISC cannot obtain the guide RNA for identifying the target transcript and thus transcript degradation cannot process properly or not at all. This protein is therefore also important for silencing a disease-causing gene by degradation of mRNA.<ref name=shuttle/><br />
<br />
<br />
<br />
<br />
== References ==<br />
<references/></div>Wally Novakhttps://proteopedia.org/index.php?title=Rde_4_sandbox&diff=2955064Rde 4 sandbox2018-10-09T04:24:29Z<p>Wally Novak: </p>
<hr />
<div>==Introduction==<br />
<br />
RDE-4 was identified through a genome-wide screening as a gene responsible for greatly reducing or abolishing RNAi in C. elegans. In organisms ranging from Arabidopsis to humans, Dicer requires dsRNA-binding proteins (dsRBPs) to carry out its roles in RNA interference (RNAi) and micro-RNA (miRNA) processing. In Caenorhabditis elegans, the dsRBP RDE-4 acts with Dicer during the initiation of RNAi, when long dsRNA is cleaved to small interfering RNAs (siRNAs). RDE-4 is not sequence-specific, however, RDE-4 binds with higher affinity to long dsRNA. Interestingly, RDE- 4 is the only protein that exhibits micromolar affinity for dsRNA in the absence of co-operativity. In addition, RDE-4 is a homodimer in solution. <ref name=first>DOI: 10.1261/rna.2338706</ref><br />
<br />
<Structure load='2LTS' size='350' frame='true' align='right' caption='Structure of RDE-4 (pdb code 2LTS)'/><br />
<br />
<Structure load='2LTR' size='350' frame='true' align='right' caption='Structure of RDE-4 (pdb code 2LTR)'/><br />
<br />
== Structure of RDE-4 ==<br />
<br />
RDE-4 has five major regions: an <scene name='79/798389/N-terminal_of_rde-4/1'>N-terminal region (residues 1-43)</scene>, two dsRBDs <scene name='79/798389/Dsrbd_residues_44-108/1'>(residues 44-108)</scene> and <scene name='79/798389/Dsrbd_residues_170-235/2'>(residues 170-235)</scene>, a <scene name='79/798389/Long_linker_of_rde-4/1'>Long Linker (residues 109-169)</scene>, and a C-terminal domain (residues 236–385). Amino acid residues 1–32 and 136–151 adopt a random coiled structure and do not make any contact within itself or with the rest of the structure. Parts of the linker, residues 109–135 and 152–169, assume an α-helix and an extended loop structure, respectively. There is not any long-range nuclear Overhauser effect information between regions 1–135 and 152– 243, suggesting that the unstructured part of the linker 136–151 separates both of these regions. The region encompassed by 236–243 folds into an α-helix. The amino acid regions 44–108 and 170–235 form dsRBD1 and dsRBD2, respectively. The hydrophobic residues Leu45, Val47, Leu48, Val55, Trp62, Met73, Leu75, Leu77, Ile80, Val82, Leu101, and Val105 stabilize dsRBD1, and Val171, Leu174, Leu183, Val201, Met205, Met227, and Leu232 form the core of dsRBD2. Apart from the canonical dsRBD fold, there are additional secondary-structural elements that are observed in both RDE-4D1 and RDE-4D2. <ref name=second>DOI: 10.1042/BJ20131347</ref> <br />
<br />
== Function ==<br />
<br />
In C. elegans, exogenous dsRNA is detected and bound by RDE-4, which stimulates dicer activity. RDE-4 initiates the siRNA pathway by binding to long dsRNA and assisting Dcr-1, a Dicer1 homologue, to facilitate siRNA production. RDE-4 interacts with Dcr-1, RDE-1, DRH-1 (Dicer-related helicase 1) and long dsRNA, which thereby suggests that RDE-4 is required only for the initiation of the RNAi pathway to generate siRNA, although high levels of dsRNA abrogate RDE-4’s role in the RNAi initiation. RDE-4 co- operatively binds to long dsRNA with nanomolar affinity, but exhibits micromolar affinity for short dsRNA, and the enhanced affinity arises from binding of several RDE-4 molecules to a single long dsRNA. <ref name=third>DOI: 10.1016/j.jmb.2008.10.002</ref> The C-terminal region of RDE-4 is necessary and sufficient to induce homodimerization RDE-4 linker– dsRBD2 are absolutely essential in C. elegans gene silencing and RDE-4 dsRBD1 and the C-terminal domain do not have any primary role in vivo. RDE-4 recognizes the A-form of nucleic acid structure adopted by dsRNA and the interaction is sequence-independent.<ref name=second/> RDE-4 dimerization is important for the assembly of active RDE-4/Dicer complexes via one of two proposed scenarios, proper Dicer recruitment to dsRNA, or facilitating Dicer dimerization. <ref name=first/> <br />
<br />
[[Image:Image-RDE-4 Mechanism.png|300px|left|thumb| A working model showing roles of RDE-1, RDE-4, and siRNAs in the interference reaction]] <ref name=fourth>DOI: 10.1017.S1355838201011074</ref><br />
<br />
<br />
== Importance ==<br />
<br />
The RDE-4 protein, and its interactions with RDE-1 and [[Dicer]], is rather important due to the fact that it cleaves the dsRNA to form small interfering RNA or microRNA, which are then integrated into the RNA-induced silencing complex. <ref name=fifth>DOI: 311/5758/195</ref> This complex then targets mRNA and prevents translation by disrupting the targeted gene. Without RDE-4, gene silencing cannot occur and without this protein, DNA and RNA cannot be regulated.<br />
<br />
== References ==<br />
<references/></div>Wally Novakhttps://proteopedia.org/index.php?title=Sandbox_RDE-1&diff=2955063Sandbox RDE-12018-10-09T04:23:37Z<p>Wally Novak: </p>
<hr />
<div>== Introduction ==<br />
<Structure load='4krf' size='350' frame='true' align='right' caption='Structure of Human Argonaute-1 let-7 complex as representation of RDE-1 since they have similar function. Thus, this complex is a model for Argonaute proteins in general.(PMID: 23746446)' scene='Insert optional scene name here' /><br />
The rde-1 gene is a member of the ''Argonaute'' gene family. Proteins from "Argonaute" family form an evolutionarily conserved family whose members silence gene expression in pathways such as RNA interference (RNAi). Argonaute family proteins can be divided into two types, AGO and Piwi proteins, depending on the small RNA they bonded to. Both types of Argonaute proteins bind 21–35 nucleotide-long small RNA guides whose sequence identifies the genes to be silenced.<ref name=four/><br />
'''RDE-1''' (RNAi-DEfective 1), a primary Argonaute protein, is required for RNA-mediated interference in ''Caenorhabditis elegans''; thus, it is also known as RNAi promoting factor. Its gene locus was first characterized in ''C. elegans'' mutants resistant to RNAi, and was found to be a member of the Piwi gene family that includes plant, Drosophila, and vertebrate homologs.<ref name=two>PMID: 10535731</ref><br />
<br />
== Structure ==<br />
All "Argonaute"/Piwi proteins possess three primary domains forming a crescent-shaped base: the PAZ, MID, and <scene name='79/798387/Piwi_domain/1'>PIWI</scene> domains. The amino-terminal PAZ domain uses its oligonucleotide-binding (OB) fold to secure the 3′ end of the small RNA guide strand to ''Argonaute'' protein. A conserved hydrophobic cavity within the PAZ domain recognizes the characteristic two-nucleotide, 3′ overhanging end of the guide-passenger siRNA generated by Dicer. The MID domain anchors the 5′ monophosphate of a siRNA to the ''Argonaute'' protein, securing the guide through multiple cycles of target cleavage. In vitro, studies suggest that 5′ phosphate binding helps align the small RNA on the surface of "Argonaute" protein, ensuring that the correct bond of the target is positioned in the endonuclease active site.<ref name=four>PMID: 21683893</ref> In other words, the PAZ and MID domains orient and anchor the double-stranded siRNA by binding to the 3’ and 5’ termini, respectively, leaving the internal nucleotides accessible for base pairing.<ref name=four/><ref name=three>PMID:15284453</ref> The carboxy-terminal PIWI domain resembles nuclease RNase H in which it folds into an RNase H-like structure. In RDE-1, this domain contains three conserved amino acids, aspartate-aspartate-histidine, that form a catalytic triad "DDH".<ref name=three/> The crystal structure of RDE-1 has not been fully elucidated, but can be assumed to closely resemble its human homologs. The full length of RDE-1 protein is 1020 amino acids (aa)<ref name=two/> in which about 110 of those aa makes up the PAZ domain and 300 aa makes up the PIWI domain.<ref> DOI: 10.1016/s0968-0004(00)01641-8</ref><br />
<br />
== Function ==<br />
[[Image:RDE-1 Proposed Fxn.png|300px||left|thumb|Proposed RNAi pathway for exogenous trigger dsRNA in C. elegans.<ref>https://en.wikipedia.org/wiki/RDE-1#/media/File:Exogenous_RNAi_Pathway_in_C._elegans,_edited.svg</ref>]] RDE-1 is not required for the initial processing of the trigger RNA into siRNAs in RNAi, but it is required in the effector step.<ref>PMID: 11680844</ref> The trigger dsRNA is bound by RDE-4 onto a [[Dicer]] complex and this complex cleaves the dsRNA into 21-25nt primary siRNA. The siRNA binds to RDE-1 promoting the formation of the RNA-induced silencing complex (RISC) and RDE-1 shuttles the siRNA to that effector complex.<ref name=promo>DOI: 10.1016/S0092-8674(02)00793-6</ref><ref name=shuttle>PMID: 12110901</ref> The RNase H activity in PIWI domain in RDE-1 facilitates siRNA maturation, cleaving siRNA into a single stranded siRNA or guide RNA<ref name=cleave>DOI: 10.1038/nsmb.1541</ref> while RISC is activated when ATP is added to the complex and utilized the guide RNA on RDE-1 to base pair with the target transcript.<ref name=shuttle/> The activation of RISC promotes the recruitment of RNA-dependent RNA polymerase (RdRP) which triggers the amplification of the secondary siRNAs to exhibit target transcript degradation.<ref>DOI: 10.1007/978-3-540-75157-1_2</ref><br />
<br />
== Importance ==<br />
Mutation of any residues in the RNase H catalytic triad abolishes Slicer activity in human Argonaute protein Ago2, suggesting that the RNase H domain is directly responsible for target mRNA degradation.<ref> PMID: 15284456</ref> However, RDE-1 has not been implicated in mRNA-cleavage activity.<br />
<br />
Instead, RDE-1 with mutations in the conserved DDH motif exhibit reduced passenger (sense) strand turnover, suggesting that RNase H activity serves to cleave the passenger strand, leaving the guide (antisense) strand accessible for base-pairing to target mRNA.<ref name=cleave/> Further, target silencing can be fully restored in DDH motif mutants by loading single-stranded siRNA, suggesting that a downstream component in the RNAi pathway is responsible for Slicer activity.<ref name=cleave/> Thus, RDE-1’s RNase H domain facilitates siRNA maturation but is not directly involved in cleaving target mRNA transcripts.<br />
<br />
RDE-1 is important for cleavage of the passenger strand and shuttling the siRNA to the appropriate RISC in ''C. elegans''.<ref name=cleave/><ref name=shuttle/> Without RDE-1, RISC cannot obtain the guide RNA for identifying the target transcript and thus transcript degradation cannot process properly or not at all. This protein is therefore also important for silencing a disease-causing gene by degradation of mRNA.<ref name=shuttle/><br />
<br />
This is a sample scene created with SAT to <scene name="/12/3456/Sample/1">color</scene> by Group, and another to make <scene name="/12/3456/Sample/2">a transparent representation</scene> of the protein. You can make your own scenes on SAT starting from scratch or loading and editing one of these sample scenes.<br />
<br />
<br />
== References ==<br />
<references/></div>Wally Novakhttps://proteopedia.org/index.php?title=Sandbox_RDE-1&diff=2955062Sandbox RDE-12018-10-09T04:22:07Z<p>Wally Novak: </p>
<hr />
<div>== Introduction ==<br />
<Structure load='4krf' size='350' frame='true' align='right' caption='Structure of Human Argonaute-1 let-7 complex as representation of RDE-1 since they have similar function. Thus, this complex is a model for Argonaute proteins in general.(PMID: 23746446)' scene='Insert optional scene name here' /><br />
The rde-1 gene is a member of the ''Argonaute'' gene family. Proteins from "Argonaute" family form an evolutionarily conserved family whose members silence gene expression in pathways such as RNA interference (RNAi). Argonaute family proteins can be divided into two types, AGO and Piwi proteins, depending on the small RNA they bonded to. Both types of Argonaute proteins bind 21–35 nucleotide-long small RNA guides whose sequence identifies the genes to be silenced.<ref name=four/><br />
'''RDE-1''' (RNAi-DEfective 1), a primary Argonaute protein, is required for RNA-mediated interference in ''Caenorhabditis elegans''; thus, it is also known as RNAi promoting factor. Its gene locus was first characterized in ''C. elegans'' mutants resistant to RNAi, and was found to be a member of the Piwi gene family that includes plant, Drosophila, and vertebrate homologs.<ref name=two>PMID: 10535731</ref><br />
<br />
== Structure ==<br />
All "Argonaute"/Piwi proteins possess three primary domains forming a crescent-shaped base: the PAZ, MID, and <scene name='79/798387/Piwi_domain/1'>PIWI</scene> domains. The amino-terminal PAZ domain uses its oligonucleotide-binding (OB) fold to secure the 3′ end of the small RNA guide strand to ''Argonaute'' protein. A conserved hydrophobic cavity within the PAZ domain recognizes the characteristic two-nucleotide, 3′ overhanging end of the guide-passenger siRNA generated by Dicer. The MID domain anchors the 5′ monophosphate of a siRNA to the ''Argonaute'' protein, securing the guide through multiple cycles of target cleavage. In vitro, studies suggest that 5′ phosphate binding helps align the small RNA on the surface of "Argonaute" protein, ensuring that the correct bond of the target is positioned in the endonuclease active site.<ref name=four>PMID: 21683893</ref> In other words, the PAZ and MID domains orient and anchor the double-stranded siRNA by binding to the 3’ and 5’ termini, respectively, leaving the internal nucleotides accessible for base pairing.<ref name=four/><ref name=three>PMID:15284453</ref> The carboxy-terminal PIWI domain resembles nuclease RNase H in which it folds into an RNase H-like structure. In RDE-1, this domain contains three conserved amino acids, aspartate-aspartate-histidine, that form a catalytic triad "DDH".<ref name=three/> The crystal structure of RDE-1 has not been fully elucidated, but can be assumed to closely resemble its human homologs. The full length of RDE-1 protein is 1020 amino acids (aa)<ref name=two/> in which about 110 of those aa makes up the PAZ domain and 300 aa makes up the PIWI domain.<ref> DOI: 10.1016/s0968-0004(00)01641-8</ref><br />
<br />
== Function ==<br />
[[Image:RDE-1 Proposed Fxn.png|300px||left|thumb|Proposed RNAi pathway for exogenous trigger dsRNA in C. elegans.<ref>https://en.wikipedia.org/wiki/RDE-1#/media/File:Exogenous_RNAi_Pathway_in_C._elegans,_edited.svg</ref>]] RDE-1 is not required for the initial processing of the trigger RNA into siRNAs in RNAi, but it is required in the effector step.<ref>PMID: 11680844</ref> The trigger dsRNA is bound by RDE-4 onto a '''Dicer''' complex and this complex cleaves the dsRNA into 21-25nt primary siRNA. The siRNA binds to RDE-1 promoting the formation of the RNA-induced silencing complex (RISC) and RDE-1 shuttles the siRNA to that effector complex.<ref name=promo>DOI: 10.1016/S0092-8674(02)00793-6</ref><ref name=shuttle>PMID: 12110901</ref> The RNase H activity in PIWI domain in RDE-1 facilitates siRNA maturation, cleaving siRNA into a single stranded siRNA or guide RNA<ref name=cleave>DOI: 10.1038/nsmb.1541</ref> while RISC is activated when ATP is added to the complex and utilized the guide RNA on RDE-1 to base pair with the target transcript.<ref name=shuttle/> The activation of RISC promotes the recruitment of RNA-dependent RNA polymerase (RdRP) which triggers the amplification of the secondary siRNAs to exhibit target transcript degradation.<ref>DOI: 10.1007/978-3-540-75157-1_2</ref><br />
<br />
== Importance ==<br />
Mutation of any residues in the RNase H catalytic triad abolishes Slicer activity in human Argonaute protein Ago2, suggesting that the RNase H domain is directly responsible for target mRNA degradation.<ref> PMID: 15284456</ref> However, RDE-1 has not been implicated in mRNA-cleavage activity.<br />
<br />
Instead, RDE-1 with mutations in the conserved DDH motif exhibit reduced passenger (sense) strand turnover, suggesting that RNase H activity serves to cleave the passenger strand, leaving the guide (antisense) strand accessible for base-pairing to target mRNA.<ref name=cleave/> Further, target silencing can be fully restored in DDH motif mutants by loading single-stranded siRNA, suggesting that a downstream component in the RNAi pathway is responsible for Slicer activity.<ref name=cleave/> Thus, RDE-1’s RNase H domain facilitates siRNA maturation but is not directly involved in cleaving target mRNA transcripts.<br />
<br />
RDE-1 is important for cleavage of the passenger strand and shuttling the siRNA to the appropriate RISC in ''C. elegans''.<ref name=cleave/><ref name=shuttle/> Without RDE-1, RISC cannot obtain the guide RNA for identifying the target transcript and thus transcript degradation cannot process properly or not at all. This protein is therefore also important for silencing a disease-causing gene by degradation of mRNA.<ref name=shuttle/><br />
<br />
This is a sample scene created with SAT to <scene name="/12/3456/Sample/1">color</scene> by Group, and another to make <scene name="/12/3456/Sample/2">a transparent representation</scene> of the protein. You can make your own scenes on SAT starting from scratch or loading and editing one of these sample scenes.<br />
<br />
<br />
== References ==<br />
<references/></div>Wally Novakhttps://proteopedia.org/index.php?title=Sandbox_RDE-1&diff=2955061Sandbox RDE-12018-10-09T04:16:00Z<p>Wally Novak: </p>
<hr />
<div>== Introduction ==<br />
<Structure load='4krf' size='350' frame='true' align='right' caption='Structure of Human Argonaute-1 let-7 complex as representation of RDE-1 since they have similar function. Thus, this complex is a model for Argonaute proteins in general.(PMID: 23746446)' scene='Insert optional scene name here' /><br />
The rde-1 gene is a member of the ''Argonaute'' gene family. Proteins from "Argonaute" family form an evolutionarily conserved family whose members silence gene expression in pathways such as RNA interference (RNAi). Argonaute family proteins can be divided into two types, AGO and Piwi proteins, depending on the small RNA they bonded to. Both types of Argonaute proteins bind 21–35 nucleotide-long small RNA guides whose sequence identifies the genes to be silenced.<ref name=four/><br />
'''RDE-1''' (RNAi-DEfective 1), a primary Argonaute protein, is required for RNA-mediated interference in ''Caenorhabditis elegans''; thus, it is also known as RNAi promoting factor. Its gene locus was first characterized in ''C. elegans'' mutants resistant to RNAi, and was found to be a member of the Piwi gene family that includes plant, Drosophila, and vertebrate homologs.<ref name=two>PMID: 10535731</ref><br />
<br />
== Structure ==<br />
All "Argonaute"/Piwi proteins possess three primary domains forming a crescent-shaped base: the PAZ, MID, and PIWI domains. The amino-terminal PAZ domain uses its oligonucleotide-binding (OB) fold to secure the 3′ end of the small RNA guide strand to ''Argonaute'' protein. A conserved hydrophobic cavity within the PAZ domain recognizes the characteristic two-nucleotide, 3′ overhanging end of the guide-passenger siRNA generated by Dicer. The MID domain anchors the 5′ monophosphate of a siRNA to the ''Argonaute'' protein, securing the guide through multiple cycles of target cleavage. In vitro, studies suggest that 5′ phosphate binding helps align the small RNA on the surface of "Argonaute" protein, ensuring that the correct bond of the target is positioned in the endonuclease active site.<ref name=four>PMID: 21683893</ref> In other words, the PAZ and MID domains orient and anchor the double-stranded siRNA by binding to the 3’ and 5’ termini, respectively, leaving the internal nucleotides accessible for base pairing.<ref name=four/><ref name=three>PMID:15284453</ref> The carboxy-terminal PIWI domain resembles nuclease RNase H in which it folds into an RNase H-like structure. In RDE-1, this domain contains three conserved amino acids, aspartate-aspartate-histidine, that form a catalytic triad "DDH".<ref name=three/> The crystal structure of RDE-1 has not been fully elucidated, but can be assumed to closely resemble its human homologs. The full length of RDE-1 protein is 1020 amino acids (aa)<ref name=two/> in which about 110 of those aa makes up the PAZ domain and 300 aa makes up the PIWI domain.<ref> DOI: 10.1016/s0968-0004(00)01641-8</ref><br />
<br />
== Function ==<br />
[[Image:RDE-1 Proposed Fxn.png|300px||left|thumb|Proposed RNAi pathway for exogenous trigger dsRNA in C. elegans.<ref>https://en.wikipedia.org/wiki/RDE-1#/media/File:Exogenous_RNAi_Pathway_in_C._elegans,_edited.svg</ref>]] RDE-1 is not required for the initial processing of the trigger RNA into siRNAs in RNAi, but it is required in the effector step.<ref>PMID: 11680844</ref> The trigger dsRNA is bound by RDE-4 onto a '''Dicer''' complex and this complex cleaves the dsRNA into 21-25nt primary siRNA. The siRNA binds to RDE-1 promoting the formation of the RNA-induced silencing complex (RISC) and RDE-1 shuttles the siRNA to that effector complex.<ref name=promo>DOI: 10.1016/S0092-8674(02)00793-6</ref><ref name=shuttle>PMID: 12110901</ref> The RNase H activity in PIWI domain in RDE-1 facilitates siRNA maturation, cleaving siRNA into a single stranded siRNA or guide RNA<ref name=cleave>DOI: 10.1038/nsmb.1541</ref> while RISC is activated when ATP is added to the complex and utilized the guide RNA on RDE-1 to base pair with the target transcript.<ref name=shuttle/> The activation of RISC promotes the recruitment of RNA-dependent RNA polymerase (RdRP) which triggers the amplification of the secondary siRNAs to exhibit target transcript degradation.<ref>DOI: 10.1007/978-3-540-75157-1_2</ref><br />
<br />
== Importance ==<br />
Mutation of any residues in the RNase H catalytic triad abolishes Slicer activity in human Argonaute protein Ago2, suggesting that the RNase H domain is directly responsible for target mRNA degradation.<ref> PMID: 15284456</ref> However, RDE-1 has not been implicated in mRNA-cleavage activity.<br />
<br />
Instead, RDE-1 with mutations in the conserved DDH motif exhibit reduced passenger (sense) strand turnover, suggesting that RNase H activity serves to cleave the passenger strand, leaving the guide (antisense) strand accessible for base-pairing to target mRNA.<ref name=cleave/> Further, target silencing can be fully restored in DDH motif mutants by loading single-stranded siRNA, suggesting that a downstream component in the RNAi pathway is responsible for Slicer activity.<ref name=cleave/> Thus, RDE-1’s RNase H domain facilitates siRNA maturation but is not directly involved in cleaving target mRNA transcripts.<br />
<br />
RDE-1 is important for cleavage of the passenger strand and shuttling the siRNA to the appropriate RISC in ''C. elegans''.<ref name=cleave/><ref name=shuttle/> Without RDE-1, RISC cannot obtain the guide RNA for identifying the target transcript and thus transcript degradation cannot process properly or not at all. This protein is therefore also important for silencing a disease-causing gene by degradation of mRNA.<ref name=shuttle/><br />
<br />
This is a sample scene created with SAT to <scene name="/12/3456/Sample/1">color</scene> by Group, and another to make <scene name="/12/3456/Sample/2">a transparent representation</scene> of the protein. You can make your own scenes on SAT starting from scratch or loading and editing one of these sample scenes.<br />
<br />
<br />
== References ==<br />
<references/></div>Wally Novakhttps://proteopedia.org/index.php?title=Sandbox_RDE-1&diff=2955060Sandbox RDE-12018-10-09T04:11:54Z<p>Wally Novak: </p>
<hr />
<div>== Introduction ==<br />
<Structure load='4krf' size='350' frame='true' align='right' caption='Structure of Human Argonaute-1 let-7 complex as representation of RDE-1 since they have similar function. Thus, this complex is a model for Argonaute proteins in general.<ref> PMID: 23746446</ref>' scene='Insert optional scene name here' /><br />
The rde-1 gene is a member of the ''Argonaute'' gene family. Proteins from "Argonaute" family form an evolutionarily conserved family whose members silence gene expression in pathways such as RNA interference (RNAi). Argonaute family proteins can be divided into two types, AGO and Piwi proteins, depending on the small RNA they bonded to. Both types of Argonaute proteins bind 21–35 nucleotide-long small RNA guides whose sequence identifies the genes to be silenced.<ref name=four/><br />
'''RDE-1''' (RNAi-DEfective 1), a primary Argonaute protein, is required for RNA-mediated interference in ''Caenorhabditis elegans''; thus, it is also known as RNAi promoting factor. Its gene locus was first characterized in ''C. elegans'' mutants resistant to RNAi, and was found to be a member of the Piwi gene family that includes plant, Drosophila, and vertebrate homologs.<ref name=two>PMID: 10535731</ref><br />
<br />
== Structure ==<br />
All "Argonaute"/Piwi proteins possess three primary domains forming a crescent-shaped base: the PAZ, MID, and PIWI domains. The amino-terminal PAZ domain uses its oligonucleotide-binding (OB) fold to secure the 3′ end of the small RNA guide strand to ''Argonaute'' protein. A conserved hydrophobic cavity within the PAZ domain recognizes the characteristic two-nucleotide, 3′ overhanging end of the guide-passenger siRNA generated by Dicer. The MID domain anchors the 5′ monophosphate of a siRNA to the ''Argonaute'' protein, securing the guide through multiple cycles of target cleavage. In vitro, studies suggest that 5′ phosphate binding helps align the small RNA on the surface of "Argonaute" protein, ensuring that the correct bond of the target is positioned in the endonuclease active site.<ref name=four>PMID: 21683893</ref> In other words, the PAZ and MID domains orient and anchor the double-stranded siRNA by binding to the 3’ and 5’ termini, respectively, leaving the internal nucleotides accessible for base pairing.<ref name=four/><ref name=three>PMID:15284453</ref> The carboxy-terminal PIWI domain resembles nuclease RNase H in which it folds into an RNase H-like structure. In RDE-1, this domain contains three conserved amino acids, aspartate-aspartate-histidine, that form a catalytic triad "DDH".<ref name=three/> The crystal structure of RDE-1 has not been fully elucidated, but can be assumed to closely resemble its human homologs. The full length of RDE-1 protein is 1020 amino acids (aa)<ref name=two/> in which about 110 of those aa makes up the PAZ domain and 300 aa makes up the PIWI domain.<ref> DOI: 10.1016/s0968-0004(00)01641-8</ref><br />
<br />
== Function ==<br />
[[Image:RDE-1 Proposed Fxn.png|300px||left|thumb|Proposed RNAi pathway for exogenous trigger dsRNA in C. elegans.<ref>https://en.wikipedia.org/wiki/RDE-1#/media/File:Exogenous_RNAi_Pathway_in_C._elegans,_edited.svg</ref>]] RDE-1 is not required for the initial processing of the trigger RNA into siRNAs in RNAi, but it is required in the effector step.<ref>PMID: 11680844</ref> The trigger dsRNA is bound by RDE-4 onto a '''Dicer''' complex and this complex cleaves the dsRNA into 21-25nt primary siRNA. The siRNA binds to RDE-1 promoting the formation of the RNA-induced silencing complex (RISC) and RDE-1 shuttles the siRNA to that effector complex.<ref name=promo>DOI: 10.1016/S0092-8674(02)00793-6</ref><ref name=shuttle>PMID: 12110901</ref> The RNase H activity in PIWI domain in RDE-1 facilitates siRNA maturation, cleaving siRNA into a single stranded siRNA or guide RNA<ref name=cleave>DOI: 10.1038/nsmb.1541</ref> while RISC is activated when ATP is added to the complex and utilized the guide RNA on RDE-1 to base pair with the target transcript.<ref name=shuttle/> The activation of RISC promotes the recruitment of RNA-dependent RNA polymerase (RdRP) which triggers the amplification of the secondary siRNAs to exhibit target transcript degradation.<ref>DOI: 10.1007/978-3-540-75157-1_2</ref><br />
<br />
== Importance ==<br />
Mutation of any residues in the RNase H catalytic triad abolishes Slicer activity in human Argonaute protein Ago2, suggesting that the RNase H domain is directly responsible for target mRNA degradation.<ref> PMID: 15284456</ref> However, RDE-1 has not been implicated in mRNA-cleavage activity.<br />
<br />
Instead, RDE-1 with mutations in the conserved DDH motif exhibit reduced passenger (sense) strand turnover, suggesting that RNase H activity serves to cleave the passenger strand, leaving the guide (antisense) strand accessible for base-pairing to target mRNA.<ref name=cleave/> Further, target silencing can be fully restored in DDH motif mutants by loading single-stranded siRNA, suggesting that a downstream component in the RNAi pathway is responsible for Slicer activity.<ref name=cleave/> Thus, RDE-1’s RNase H domain facilitates siRNA maturation but is not directly involved in cleaving target mRNA transcripts.<br />
<br />
RDE-1 is important for cleavage of the passenger strand and shuttling the siRNA to the appropriate RISC in ''C. elegans''.<ref name=cleave/><ref name=shuttle/> Without RDE-1, RISC cannot obtain the guide RNA for identifying the target transcript and thus transcript degradation cannot process properly or not at all. This protein is therefore also important for silencing a disease-causing gene by degradation of mRNA.<ref name=shuttle/><br />
<br />
This is a sample scene created with SAT to <scene name="/12/3456/Sample/1">color</scene> by Group, and another to make <scene name="/12/3456/Sample/2">a transparent representation</scene> of the protein. You can make your own scenes on SAT starting from scratch or loading and editing one of these sample scenes.<br />
<br />
<br />
== References ==<br />
<references/></div>Wally Novakhttps://proteopedia.org/index.php?title=Sandbox_RDE-1&diff=2955059Sandbox RDE-12018-10-09T04:02:33Z<p>Wally Novak: </p>
<hr />
<div>== Introduction ==<br />
<Structure load='4krf' size='350' frame='true' align='right' caption='Protein length: 1020 aa, Mass: 118,804 Da' scene='Insert optional scene name here' /><br />
The rde-1 gene is a member of the ''Argonaute'' gene family. Proteins from "Argonaute" family form an evolutionarily conserved family whose members silence gene expression in pathways such as RNA interference (RNAi). Argonaute family proteins can be divided into two types, AGO and Piwi proteins, depending on the small RNA they bonded to. Both types of Argonaute proteins bind 21–35 nucleotide-long small RNA guides whose sequence identifies the genes to be silenced.<ref name=four/><br />
'''RDE-1''' (RNAi-DEfective 1), a primary Argonaute protein, is required for RNA-mediated interference in ''Caenorhabditis elegans''; thus, it is also known as RNAi promoting factor. Its gene locus was first characterized in ''C. elegans'' mutants resistant to RNAi, and was found to be a member of the Piwi gene family that includes plant, Drosophila, and vertebrate homologs.<ref name=two>PMID: 10535731</ref><br />
<br />
== Structure ==<br />
All "Argonaute"/Piwi proteins possess three primary domains forming a crescent-shaped base: the PAZ, MID, and PIWI domains. The amino-terminal PAZ domain uses its oligonucleotide-binding (OB) fold to secure the 3′ end of the small RNA guide strand to ''Argonaute'' protein. A conserved hydrophobic cavity within the PAZ domain recognizes the characteristic two-nucleotide, 3′ overhanging end of the guide-passenger siRNA generated by Dicer. The MID domain anchors the 5′ monophosphate of a siRNA to the ''Argonaute'' protein, securing the guide through multiple cycles of target cleavage. In vitro, studies suggest that 5′ phosphate binding helps align the small RNA on the surface of "Argonaute" protein, ensuring that the correct bond of the target is positioned in the endonuclease active site.<ref name=four>PMID: 21683893</ref> In other words, the PAZ and MID domains orient and anchor the double-stranded siRNA by binding to the 3’ and 5’ termini, respectively, leaving the internal nucleotides accessible for base pairing.<ref name=four/><ref name=three>PMID:15284453</ref> The carboxy-terminal PIWI domain resembles nuclease RNase H in which it folds into an RNase H-like structure. In RDE-1, this domain contains three conserved amino acids, aspartate-aspartate-histidine, that form a catalytic triad "DDH".<ref name=three/> The crystal structure of RDE-1 has not been fully elucidated, but can be assumed to closely resemble its human homologs. The full length of RDE-1 protein is 1020 amino acids (aa)<ref name=two/> in which about 110 of those aa makes up the PAZ domain and 300 aa makes up the PIWI domain.<ref> DOI: 10.1016/s0968-0004(00)01641-8</ref><br />
<br />
== Function ==<br />
[[Image:RDE-1 Proposed Fxn.png|300px||left|thumb|Proposed RNAi pathway for exogenous trigger dsRNA in C. elegans.<ref>https://en.wikipedia.org/wiki/RDE-1#/media/File:Exogenous_RNAi_Pathway_in_C._elegans,_edited.svg</ref>]] '''RDE-1''' is not required for the initial processing of the trigger RNA into siRNAs in RNAi, but it is required in the effector step.<ref>PMID: 11680844</ref> The trigger dsRNA is bound by RDE-4 onto a '''Dicer''' complex and this complex cleaves the dsRNA into 21-25nt primary siRNA. The siRNA binds to RDE-1 promoting the formation of the RNA-induced silencing complex (RISC) and RDE-1 shuttles the siRNA to that effector complex.<ref name=promo>DOI: 10.1016/S0092-8674(02)00793-6</ref><ref name=shuttle>PMID: 12110901</ref> The RNase H activity in PIWI domain in RDE-1 facilitates siRNA maturation, cleaving siRNA into a single stranded siRNA or guide RNA<ref name=cleave>DOI: 10.1038/nsmb.1541</ref> while RISC is activated when ATP is added to the complex and utilized the guide RNA on RDE-1 to base pair with the target transcript.<ref name=shuttle/> The activation of RISC promotes the recruitment of RNA-dependent RNA polymerase (RdRP) which triggers the amplification of the secondary siRNAs to exhibit target transcript degradation.<ref>DOI: 10.1007/978-3-540-75157-1_2</ref><br />
<br />
== Importance ==<br />
Mutation of any residues in the RNase H catalytic triad abolishes Slicer activity in human Argonaute protein Ago2, suggesting that the RNase H domain is directly responsible for target mRNA degradation.<ref> PMID: 15284456</ref> However, RDE-1 has not been implicated in mRNA-cleavage activity.<br />
<br />
Instead, RDE-1 with mutations in the conserved DDH motif exhibit reduced passenger (sense) strand turnover, suggesting that RNase H activity serves to cleave the passenger strand, leaving the guide (antisense) strand accessible for base-pairing to target mRNA.<ref name=cleave/> Further, target silencing can be fully restored in DDH motif mutants by loading single-stranded siRNA, suggesting that a downstream component in the RNAi pathway is responsible for Slicer activity.<ref name=cleave/> Thus, RDE-1’s RNase H domain facilitates siRNA maturation but is not directly involved in cleaving target mRNA transcripts.<br />
<br />
RDE-1 is important for cleavage of the passenger strand and shuttling the siRNA to the appropriate RISC in ''C. elegans''.<ref name=cleave/><ref name=shuttle/> Without RDE-1, RISC cannot obtain the guide RNA for identifying the target transcript and thus transcript degradation cannot process properly or not at all. This protein is therefore also important for silencing a disease-causing gene by degradation of mRNA.<ref name=shuttle/><br />
<br />
This is a sample scene created with SAT to <scene name="/12/3456/Sample/1">color</scene> by Group, and another to make <scene name="/12/3456/Sample/2">a transparent representation</scene> of the protein. You can make your own scenes on SAT starting from scratch or loading and editing one of these sample scenes.<br />
<br />
<br />
== References ==<br />
<references/></div>Wally Novakhttps://proteopedia.org/index.php?title=Sandbox_RDE-1&diff=2955058Sandbox RDE-12018-10-09T03:54:02Z<p>Wally Novak: </p>
<hr />
<div>== Introduction ==<br />
<Structure load='4krf' size='350' frame='true' align='right' caption='Protein length: 1020 aa, Mass: 118,804 Da' scene='Insert optional scene name here' /><br />
The rde-1 gene is a member of the ''Argonaute'' gene family. Proteins from "Argonaute" family form an evolutionarily conserved family whose members silence gene expression in pathways such as RNA interference (RNAi). Argonaute family proteins can be divided into two types, AGO and Piwi proteins, depending on the small RNA they bonded to. Both types of Argonaute proteins bind 21–35 nucleotide-long small RNA guides whose sequence identifies the genes to be silenced.<ref name=four/><br />
'''RDE-1''' (RNAi-DEfective 1), a primary Argonaute protein, is required for RNA-mediated interference in ''Caenorhabditis elegans''; thus, it is also known as RNAi promoting factor. Its gene locus was first characterized in ''C. elegans'' mutants resistant to RNAi, and was found to be a member of the Piwi gene family that includes plant, Drosophila, and vertebrate homologs.<ref name=two>PMID: 10535731</ref><br />
<br />
== Structure ==<br />
All "Argonaute"/Piwi proteins possess three primary domains forming a crescent-shaped base: the PAZ, MID, and PIWI domains. The amino-terminal PAZ domain uses its oligonucleotide-binding (OB) fold to secure the 3′ end of the small RNA guide strand to ''Argonaute'' protein. A conserved hydrophobic cavity within the PAZ domain recognizes the characteristic two-nucleotide, 3′ overhanging end of the guide-passenger siRNA generated by Dicer. The MID domain anchors the 5′ monophosphate of a siRNA to the ''Argonaute'' protein, securing the guide through multiple cycles of target cleavage. In vitro, studies suggest that 5′ phosphate binding helps align the small RNA on the surface of "Argonaute" protein, ensuring that the correct bond of the target is positioned in the endonuclease active site.<ref name=four>PMID: 21683893</ref> In other words, the PAZ and MID domains orient and anchor the double-stranded siRNA by binding to the 3’ and 5’ termini, respectively, leaving the internal nucleotides accessible for base pairing.<ref name=four/><ref name=three>PMID:15284453</ref> The carboxy-terminal PIWI domain resembles nuclease RNase H in which it folds into an RNase H-like structure. In RDE-1, this domain contains three conserved amino acids, aspartate-aspartate-histidine, that form a catalytic triad "DDH".<ref name=three/> The crystal structure of RDE-1 has not been fully elucidated, but can be assumed to closely resemble its human homologs. The full length of RDE-1 protein is 1020 amino acids (aa)<ref name=two/> in which about 110 of those aa makes up the PAZ domain and 300 aa makes up the PIWI domain.<ref> DOI: 10.1016/s0968-0004(00)01641-8</ref><br />
<br />
== Function ==<br />
[[Image:RDE-1 Proposed Fxn.png|300px||left|thumb|Proposed RNAi pathway for exogenous trigger dsRNA in C. elegans.<ref>https://en.wikipedia.org/wiki/RDE-1#/media/File:Exogenous_RNAi_Pathway_in_C._elegans,_edited.svg</ref>]] '''RDE-1''' is not required for the initial processing of the trigger RNA into siRNAs in RNAi, but it is required in the effector step.<ref>PMID: 11680844</ref> The trigger dsRNA is bound by RDE-4 onto a '''Dicer''' complex and this complex cleaves the dsRNA into 21-25nt primary siRNA. The siRNA binds to RDE-1 promoting the formation of the RNA-induced silencing complex (RISC) and RDE-1 shuttles the siRNA to that effector complex.<ref name=promo>DOI: 10.1016/S0092-8674(02)00793-6</ref><ref name=shuttle>PMID: 12110901</ref> The RNase H activity in PIWI domain in RDE-1 facilitates siRNA maturation, cleaving siRNA into a single stranded siRNA or guide RNA<ref> DOI: 10.1038/nsmb.1541</ref> while RISC is activated when ATP is added to the complex and utilized the guide RNA on RDE-1 to base pair with the target transcript.<ref name=shuttle/> The activation of RISC promotes the recruitment of RNA-dependent RNA polymerase (RdRP) which triggers the amplification of the secondary siRNAs to exhibit target transcript degradation.<ref>DOI: 10.1007/978-3-540-75157-1_2</ref><br />
<br />
== Importance ==<br />
Mutation of any residues in the RNase H catalytic triad abolishes Slicer activity in human Argonaute protein Ago2, suggesting that the RNase H domain is directly responsible for target mRNA degradation.[10] However, RDE-1 has not been implicated in mRNA-cleavage activity.<br />
<br />
Instead, RDE-1 with mutations in the conserved DDH motif exhibit reduced passenger (sense) strand turnover, suggesting that RNase H activity serves to cleave the passenger strand, leaving the guide (antisense) strand accessible for base-pairing to target mRNA.[5] Further, target silencing can be fully restored in DDH motif mutants by loading single-stranded siRNA, suggesting that a downstream component in the RNAi pathway is responsible for Slicer activity.[5] Thus, RDE-1’s RNase H domain facilitates siRNA maturation but is not directly involved in cleaving target mRNA transcripts.<br />
<br />
RDE-1 is important for cleavage of the passenger strand and shuttling the siRNA to the appropriate RISC in ''C. elegans''. Without RDE-1, RISC cannot obtain the guide RNA for identifying the target transcript and thus transcript degradation cannot process properly or not at all. This protein is therefore also important for silencing a disease-causing gene by degradation of mRNA.<br />
<br />
This is a sample scene created with SAT to <scene name="/12/3456/Sample/1">color</scene> by Group, and another to make <scene name="/12/3456/Sample/2">a transparent representation</scene> of the protein. You can make your own scenes on SAT starting from scratch or loading and editing one of these sample scenes.<br />
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<br />
== References ==<br />
<references/></div>Wally Novakhttps://proteopedia.org/index.php?title=Rde_4_sandbox&diff=2955057Rde 4 sandbox2018-10-09T03:37:32Z<p>Wally Novak: </p>
<hr />
<div>==Introduction==<br />
<br />
RDE-4 was identified through a genome-wide screening as a gene responsible for greatly reducing or abolishing RNAi in C. elegans. In organisms ranging from Arabidopsis to humans, Dicer requires dsRNA-binding proteins (dsRBPs) to carry out its roles in RNA interference (RNAi) and micro-RNA (miRNA) processing. In Caenorhabditis elegans, the dsRBP RDE-4 acts with Dicer during the initiation of RNAi, when long dsRNA is cleaved to small interfering RNAs (siRNAs). RDE-4 is not sequence-specific, however, RDE-4 binds with higher affinity to long dsRNA. Interestingly, RDE- 4 is the only protein that exhibits micromolar affinity for dsRNA in the absence of co-operativity. In addition, RDE-4 is a homodimer in solution. <ref name=first>DOI: 10.1261/rna.2338706</ref><br />
<br />
<Structure load='2LTS' size='350' frame='true' align='right' caption='Structure of RDE-4 (pdb code 2LTS)'/><br />
<br />
<Structure load='2LTR' size='350' frame='true' align='right' caption='Structure of RDE-4 (pdb code 2LTR)'/><br />
<br />
== Structure of RDE-4 ==<br />
<br />
RDE-4 has five major regions: an <scene name='79/798389/N-terminal_of_rde-4/1'>N-terminal region (residues 1-43)</scene>, two dsRBDs <scene name='79/798389/Dsrbd_residues_44-108/1'>(residues 44-108)</scene> and <scene name='79/798389/Dsrbd_residues_170-235/2'>(residues 170-235)</scene>, a <scene name='79/798389/Long_linker_of_rde-4/1'>Long Linker (residues 109-169)</scene>, and a C-terminal domain (residues 236–385). Amino acid residues 1–32 and 136–151 adopt a random coiled structure and do not make any contact within itself or with the rest of the structure. Parts of the linker, residues 109–135 and 152–169, assume an α-helix and an extended loop structure, respectively. There is not any long-range nuclear Overhauser effect information between regions 1–135 and 152– 243, suggesting that the unstructured part of the linker 136–151 separates both of these regions. The region encompassed by 236–243 folds into an α-helix. The amino acid regions 44–108 and 170–235 form dsRBD1 and dsRBD2, respectively. The hydrophobic residues Leu45, Val47, Leu48, Val55, Trp62, Met73, Leu75, Leu77, Ile80, Val82, Leu101, and Val105 stabilize dsRBD1, and Val171, Leu174, Leu183, Val201, Met205, Met227, and Leu232 form the core of dsRBD2. Apart from the canonical dsRBD fold, there are additional secondary-structural elements that are observed in both RDE-4D1 and RDE-4D2. <ref name=second>DOI: 10.1042/BJ20131347</ref> <br />
<br />
== Function ==<br />
<br />
In C. elegans, exogenous dsRNA is detected and bound by RDE-4, which stimulates dicer activity. RDE-4 initiates the siRNA pathway by binding to long dsRNA and assisting Dcr-1, a Dicer1 homologue, to facilitate siRNA production. RDE-4 interacts with Dcr-1, RDE-1, DRH-1 (Dicer-related helicase 1) and long dsRNA, which thereby suggests that RDE-4 is required only for the initiation of the RNAi pathway to generate siRNA, although high levels of dsRNA abrogate RDE-4’s role in the RNAi initiation. RDE-4 co- operatively binds to long dsRNA with nanomolar affinity, but exhibits micromolar affinity for short dsRNA, and the enhanced affinity arises from binding of several RDE-4 molecules to a single long dsRNA. <ref name=third>DOI: 10.1016/j.jmb.2008.10.002</ref> The C-terminal region of RDE-4 is necessary and sufficient to induce homodimerization RDE-4 linker– dsRBD2 are absolutely essential in C. elegans gene silencing and RDE-4 dsRBD1 and the C-terminal domain do not have any primary role in vivo. RDE-4 recognizes the A-form of nucleic acid structure adopted by dsRNA and the interaction is sequence-independent.<ref name=second/> RDE-4 dimerization is important for the assembly of active RDE-4/Dicer complexes via one of two proposed scenarios, proper Dicer recruitment to dsRNA, or facilitating Dicer dimerization. <ref name=first/> <br />
<br />
[[Image:Image-RDE-4 Mechanism.png|300px|left|thumb| A working model showing roles of RDE-1, RDE-4, and siRNAs in the interference reaction]] <ref name=fourth>DOI: 10.1017.S1355838201011074</ref><br />
<br />
<br />
== Importance ==<br />
<br />
The RDE-4 protein, and its interactions with RDE-1 and Dicer, is rather important due to the fact that it cleaves the dsRNA to form small interfering RNA or microRNA, which are then integrated into the RNA-induced silencing complex. <ref name=fifth>DOI: 311/5758/195</ref> This complex then targets mRNA and prevents translation by disrupting the targeted gene. Without RDE-4, gene silencing cannot occur and without this protein, DNA and RNA cannot be regulated.<br />
<br />
== References ==<br />
<references/></div>Wally Novakhttps://proteopedia.org/index.php?title=Rde_4_sandbox&diff=2955056Rde 4 sandbox2018-10-09T03:28:51Z<p>Wally Novak: </p>
<hr />
<div>==Introduction==<br />
<br />
RDE-4 was identified through a genome-wide screening as a gene responsible for greatly reducing or abolishing RNAi in C. elegans. In organisms ranging from Arabidopsis to humans, Dicer requires dsRNA-binding proteins (dsRBPs) to carry out its roles in RNA interference (RNAi) and micro-RNA (miRNA) processing. In Caenorhabditis elegans, the dsRBP RDE-4 acts with Dicer during the initiation of RNAi, when long dsRNA is cleaved to small interfering RNAs (siRNAs). RDE-4 is not sequence-specific, however, RDE-4 binds with higher affinity to long dsRNA. Interestingly, RDE- 4 is the only protein that exhibits micromolar affinity for dsRNA in the absence of co-operativity. In addition, RDE-4 is a homodimer in solution. <ref name=first>DOI: 10.1261/rna.2338706</ref><br />
<br />
<Structure load='2LTS' size='350' frame='true' align='right' caption='Structure of RDE-4 (pdb code 2LTS)'/><br />
<br />
<Structure load='2LTR' size='350' frame='true' align='right' caption='Structure of RDE-4 (pdb code 2LTR)'/><br />
<br />
== Structure of RDE-4 ==<br />
<br />
RDE-4 has five major regions: an <scene name='79/798389/N-terminal_of_rde-4/1'>N-terminal region (residues 1-43)</scene>, two dsRBDs <scene name='79/798389/Dsrbd_residues_44-108/1'>(residues 44-108)</scene> and <scene name='79/798389/Dsrbd_residues_170-235/2'>(residues 170-235)</scene>, a <scene name='79/798389/Long_linker_of_rde-4/1'>Long Linker (residues 109-169)</scene>, and a C-terminal domain (residues 236–385). Amino acid residues 1–32 and 136–151 adopt a random coiled structure and do not make any contact within itself or with the rest of the structure. Parts of the linker, residues 109–135 and 152–169, assume an α-helix and an extended loop structure, respectively. There is not any long-range nuclear Overhauser effect information between regions 1–135 and 152– 243, suggesting that the unstructured part of the linker 136–151 separates both of these regions. The region encompassed by 236–243 folds into an α-helix. The amino acid regions 44–108 and 170–235 form dsRBD1 and dsRBD2, respectively. The hydrophobic residues Leu45, Val47, Leu48, Val55, Trp62, Met73, Leu75, Leu77, Ile80, Val82, Leu101, and Val105 stabilize dsRBD1, and Val171, Leu174, Leu183, Val201, Met205, Met227, and Leu232 form the core of dsRBD2. Apart from the canonical dsRBD fold, there are additional secondary-structural elements that are observed in both RDE-4D1 and RDE-4D2. <ref name=second>DOI: 10.1042/BJ20131347</ref> <br />
<br />
== Function ==<br />
<br />
In C. elegans, exogenous dsRNA is detected and bound by RDE-4, which stimulates dicer activity. RDE-4 initiates the siRNA pathway by binding to long dsRNA and assisting Dcr-1, a Dicer1 homologue, to facilitate siRNA production. RDE-4 interacts with Dcr-1, RDE-1, DRH-1 (Dicer-related helicase 1) and long dsRNA, which thereby suggests that RDE-4 is required only for the initiation of the RNAi pathway to generate siRNA, although high levels of dsRNA abrogate RDE-4’s role in the RNAi initiation. RDE-4 co- operatively binds to long dsRNA with nanomolar affinity, but exhibits micromolar affinity for short dsRNA, and the enhanced affinity arises from binding of several RDE-4 molecules to a single long dsRNA. <ref name=third>DOI: 10.1016/j.jmb.2008.10.002</ref> The C-terminal region of RDE-4 is necessary and sufficient to induce homodimerization RDE-4 linker– dsRBD2 are absolutely essential in C. elegans gene silencing and RDE-4 dsRBD1 and the C-terminal domain do not have any primary role in vivo. RDE-4 recognizes the A-form of nucleic acid structure adopted by dsRNA and the interaction is sequence-independent.<ref name=second/> RDE-4 dimerization is important for the assembly of active RDE-4/Dicer complexes via one of two proposed scenarios, proper Dicer recruitment to dsRNA, or facilitating Dicer dimerization. <ref name=first/> <br />
<br />
[[Image:Image-RDE-4 Mechanism.png|300px|left|thumb| A working model showing roles of RDE-1, RDE-4, and siRNAs in the interference reaction]] <ref name=fourth>DOI: 10.1017.S1355838201011074</ref><br />
<br />
<br />
== Importance ==<br />
<br />
The RDE-4 protein, and its interactions with RDE-1 and Dicer, is rather important due to the fact that it cleaves the dsRNA to form small interfering RNA or <br />
<br />
== References ==<br />
<references/></div>Wally Novakhttps://proteopedia.org/index.php?title=User:Wally_Novak/Sandbox_Whitaker&diff=2955055User:Wally Novak/Sandbox Whitaker2018-10-09T03:24:00Z<p>Wally Novak: </p>
<hr />
<div>==U5 snRNP==<br />
<StructureSection load='1stp' size='340' side='right' caption='Human Spliceosomal Protein U5-15kD'><br />
==Function==<br />
===General Overview===<br />
The U5 snRNP consists of several proteins which are highly conserved from yeast to humans. These proteins consist of <scene name='79/798410/Yeast_prp8/1'>Prp8</scene>, <scene name='79/798410/Snu114/1'>Snu114</scene>, <scene name='79/798410/Yeast_brr2/1'>Brr2</scene>, Prp28, Snu40/52K, and <scene name='79/798410/Dib1/1'>Dib1</scene><ref>PMID:11720284</ref>, however, only Prp8, Snu114, and Brr2 remain in complex with the U5 snRNA while the spliceosome is activated <ref name="Irish">PMID:23354046</ref>. These proteins are necessary for the remodeling of the spliceosome and may play a role in maintaining catalytic activity at the active center of the spliceosome <ref name="Irish" />. Assembly of the U5 snRNP is followed by the formation of the U4/U6.U5 <scene name='79/798410/Prp8_snu114_brr2_5gan/1'>tri-snRNP</scene> which is the last remaining complex necessary to form the complete spliceosome and is therefore vital for its function. <br />
===U5 snRNA===<br />
The U5 snRNA interacts directly with the 5’ exon before the first step of splicing and with the 5’ and 3’ exons following the first step of splicing <ref name="Keefe">PMID:9430647</ref>. These U5–exon interactions are essential for tethering and aligning the exons for ligation during the second step of splicing <ref name="Keefe" />.<br />
===Brr2===<br />
<Structure load='3hib' size='400' frame='true' align='right' caption='Brr2="/><br />
Brr2 is an ATPase required for unwinding U4/U6 base-pairing before the first step of splicing and for unwinding U2/U6 base-pairing after the second step of splicing <ref>PMID:20659012</ref>. These conformational changes catalyzed by Brr2 are essential for the progression of the spliceosome cycle. <br />
===Prp8===<br />
<Structure load='3e66' size='400' frame='true' align='right' caption='Prp8'/><br />
Prp8 is believed to be the master regulator of the splicing cycle by regulating the spliceosomal DExD/H-box RNA dependent ATPases, specifically Brr2 <ref>PMID:11017191</ref>. Consistent with this regulatory role it has been shown that the C-terminus of Prp8 activates Brr2 helicase function and inhibits Brr2's U4/U6-dependent ATPase activity in vitro <ref>PMID:19098916</ref>. Structural studies of the Prp8 C-terminus have identified an RNase H-like domain within Prp8 and it has been proposed that this RNase H-domain may form the active site of the spliceosome <ref name="Irish" />.<br />
===Snu114===<br />
<scene name='79/798410/Snu114/1'>Snu114</scene> is the only GTPase associated with the spliceosome and it has multiple contacts with Prp8 as well as U5 snRNA and Brr2 but its role in spliceosome function is not yet well-defined. <ref name="Irish" />.<br />
==Structural Highlights==<br />
Four sequences are highly conserved within the U5 snRNA and it is believed that these sequences play a primary role in the proteins binding capabilities. The four regions are the <scene name='79/798410/Stem_loop/1'>Stem-loop 1</scene>, Internal loop 1, Stem-closing internal loop 1, and Sm protein binding site <ref name="Frank">PMID:8114748</ref>. <scene name='79/798410/Stem_loop/1'>Stem-loop 1</scene> spans nucleotides 84 to 110 (S. cerevisiae) and consists of a highly conserved 11 nucleotide block with complementary sequences on either side <ref name="Frank" />. These complementary sequences enable local base-pairing to form the stem of the loop <ref name="Frank" />. The U5 snRNA stem-loop 1 is necessary for tethering and aligning exons for ligation during the second step of splicing. The U5 snRNA <scene name='79/798410/Stem_loop/1'>stem 1</scene> and internal loop 1 (IL1) are also highly conserved between yeast and humans, both in size and in structure <ref name="Frank" />. The U5 snRNA IL1 was found to be the most important region for the association of Prp8, Snu114 and Brr2 with the U5 snRNA <ref>PMID:23857713</ref>. Genetic analysis also identified the U5 snRNA IL1 as being important for Brr2 function <ref name="Frank" />. Overall, the U5 snRNA IL1 appears to be critical for the association of the U5 snRNP proteins Prp8, Snu114 and Brr2 with the U5 snRNA to form the <scene name='79/798410/Prp8_snu114_brr2_5gan/2'>U5 snRNP</scene> <ref name="Frank" />. <br />
===Brr2 Structural Highlights===<br />
<Structure load='3hib' size='400' frame='true' align='right' caption='Brr2'/><br />
Recent structural analysis of Brr2 has revealed that parts of the Sec63 like cassettes of the Brr2 helicase domains resembles the DNA helicase Hel308, hinting at an RNA unwinding action of Brr2 similar to that of the DNA unwinding by helicase Hel308 <ref name="Irish" />. The activity of Brr2 is regulated by the guanine nucleotide state of Snu114 <ref name="Irish" />.<br />
===Prp8 Structural Highlights===<br />
<Structure load='3e66' size='400' frame='true' align='right' caption='Prp8'/><br />
Prp8 is a 280 kDa protein component of the U5 snRNP and is also part of the U4/U6.U5 tri-snRNP <ref name="Irish" />. Prp8 forms a complex with the assembly factor Aar2 and Snu114 without the U5 snRNA in the cytoplasm, however, Aar2 is replaced with Brr2 in the nucleus <ref name="Irish" />.<br />
==Protein Interactions==<br />
The U5 snRNP is one of the five subunits which come together to form the spliceosome and is, therefore, a key protein for eukaryotic mRNA processing. U5 snRNP is a complex which consists of U5 snRNA, Brr2, Snu114, and Prp8. Prp8 also interacts with other proteins of the spliceosome, the snRNAs and extensively with the pre-mRNA <ref name="Irish" />. It has been shown that Prp8 crosslinks directly to the pre-mRNA 5’ splice site, the branch site, and the 3’ splice site as well as U5 and U6 snRNAs localizing it to the heart of the spliceosome <ref name="Irish" />. In yeast, Prp8 makes extensive direct contacts with the U5 snRNA including the highly conserved U5 loop 1 and IL1 <ref name="Irish" />.<br />
Following successful assembly of the U5 snRNP, a <scene name='79/798410/Prp8_snu114_brr2_5gan/1'>tri-snRNP</scene> consisting of U4, U5 and U6 is formed which is then incorporated into the active spliceosome. <Structure load='5gan' size='400' frame='true' align='right' caption='U4/U6.U5 tri-snRNP'/><br />
<br />
<br />
</StructureSection><br />
== References ==<br />
<references/></div>Wally Novakhttps://proteopedia.org/index.php?title=File:Image-RDE-4_Mechanism.png&diff=2955054File:Image-RDE-4 Mechanism.png2018-10-09T03:22:57Z<p>Wally Novak: </p>
<hr />
<div></div>Wally Novakhttps://proteopedia.org/index.php?title=File:RDE-4_Mechanism.png&diff=2955053File:RDE-4 Mechanism.png2018-10-09T03:18:33Z<p>Wally Novak: </p>
<hr />
<div></div>Wally Novakhttps://proteopedia.org/index.php?title=Rde_4_sandbox&diff=2955052Rde 4 sandbox2018-10-09T03:17:50Z<p>Wally Novak: </p>
<hr />
<div>==Introduction==<br />
<br />
RDE-4 was identified through a genome-wide screening as a gene responsible for greatly reducing or abolishing RNAi in C. elegans. In organisms ranging from Arabidopsis to humans, Dicer requires dsRNA-binding proteins (dsRBPs) to carry out its roles in RNA interference (RNAi) and micro-RNA (miRNA) processing. In Caenorhabditis elegans, the dsRBP RDE-4 acts with Dicer during the initiation of RNAi, when long dsRNA is cleaved to small interfering RNAs (siRNAs). RDE-4 is not sequence-specific, however, RDE-4 binds with higher affinity to long dsRNA. Interestingly, RDE- 4 is the only protein that exhibits micromolar affinity for dsRNA in the absence of co-operativity. In addition, RDE-4 is a homodimer in solution. <ref name=first>DOI: 10.1261/rna.2338706</ref><br />
<br />
<Structure load='2LTS' size='350' frame='true' align='right' caption='Structure of RDE-4 (pdb code 2LTS)'/><br />
<br />
<Structure load='2LTR' size='350' frame='true' align='right' caption='Structure of RDE-4 (pdb code 2LTR)'/><br />
<br />
== Structure of RDE-4 ==<br />
<br />
RDE-4 has five major regions: an <scene name='79/798389/N-terminal_of_rde-4/1'>N-terminal region (residues 1-43)</scene>, two dsRBDs <scene name='79/798389/Dsrbd_residues_44-108/1'>(residues 44-108)</scene> and <scene name='79/798389/Dsrbd_residues_170-235/2'>(residues 170-235)</scene>, a <scene name='79/798389/Long_linker_of_rde-4/1'>Long Linker (residues 109-169)</scene>, and a C-terminal domain (residues 236–385). Amino acid residues 1–32 and 136–151 adopt a random coiled structure and do not make any contact within itself or with the rest of the structure. Parts of the linker, residues 109–135 and 152–169, assume an α-helix and an extended loop structure, respectively. There is not any long-range nuclear Overhauser effect information between regions 1–135 and 152– 243, suggesting that the unstructured part of the linker 136–151 separates both of these regions. The region encompassed by 236–243 folds into an α-helix. The amino acid regions 44–108 and 170–235 form dsRBD1 and dsRBD2, respectively. The hydrophobic residues Leu45, Val47, Leu48, Val55, Trp62, Met73, Leu75, Leu77, Ile80, Val82, Leu101, and Val105 stabilize dsRBD1, and Val171, Leu174, Leu183, Val201, Met205, Met227, and Leu232 form the core of dsRBD2. Apart from the canonical dsRBD fold, there are additional secondary-structural elements that are observed in both RDE-4D1 and RDE-4D2. <ref name=second>DOI:DOI: 10.1042/BJ20131347</ref> <br />
<br />
== Function ==<br />
<br />
In C. elegans, exogenous dsRNA is detected and bound by RDE-4, which stimulates dicer activity. RDE-4 initiates the siRNA pathway by binding to long dsRNA and assisting Dcr-1, a Dicer1 homologue, to facilitate siRNA production. RDE-4 interacts with Dcr-1, RDE-1, DRH-1 (Dicer-related helicase 1) and long dsRNA, which thereby suggests that RDE-4 is required only for the initiation of the RNAi pathway to generate siRNA, although high levels of dsRNA abrogate RDE-4’s role in the RNAi initiation. RDE-4 co- operatively binds to long dsRNA with nanomolar affinity, but exhibits micromolar affinity for short dsRNA, and the enhanced affinity arises from binding of several RDE-4 molecules to a single long dsRNA. <ref name=third>DOI: 10.1016/j.jmb.2008.10.002</ref> The C-terminal region of RDE-4 is necessary and sufficient to induce homodimerization RDE-4 linker– dsRBD2 are absolutely essential in C. elegans gene silencing and RDE-4 dsRBD1 and the C-terminal domain do not have any primary role in vivo. RDE-4 recognizes the A-form of nucleic acid structure adopted by dsRNA and the interaction is sequence-independent.<ref name=second/> RDE-4 dimerization is important for the assembly of active RDE-4/Dicer complexes via one of two proposed scenarios, proper Dicer recruitment to dsRNA, or facilitating Dicer dimerization. <ref name=first/> <br />
<br />
== Importance ==<br />
<br />
The RDE-4 protein, and its interactions with RDE-1 and Dicer, is rather important due to the fact that it cleaves the dsRNA to form small interfering RNA or <br />
<br />
== References ==<br />
<references/></div>Wally Novakhttps://proteopedia.org/index.php?title=User:Wally_Novak/Sandbox_Whitaker&diff=2955051User:Wally Novak/Sandbox Whitaker2018-10-09T03:09:13Z<p>Wally Novak: </p>
<hr />
<div>==U5 snRNP==<br />
<StructureSection load='1stp' size='340' side='right' caption='Human Spliceosomal Protein U5-15kD'><br />
==Function==<br />
===General Overview===<br />
The U5 snRNP consists of several proteins which are highly conserved from yeast to humans. These proteins consist of <scene name='79/798410/Yeast_prp8/1'>Prp8</scene>, <scene name='79/798410/Snu114/1'>Snu114</scene>, <scene name='79/798410/Yeast_brr2/1'>Brr2</scene>, Prp28, Snu40/52K, and <scene name='79/798410/Dib1/1'>Dib1</scene><ref>PMID:11720284</ref>, however, only Prp8, Snu114, and Brr2 remain in complex with the U5 snRNA while the spliceosome is activated <ref name="Irish">PMID:23354046</ref>. These proteins are necessary for the remodeling of the spliceosome and may play a role in maintaining catalytic activity at the active center of the spliceosome <ref name="Irish" />. Assembly of the U5 snRNP is followed by the formation of the U4/U6.U5 <scene name='79/798410/Prp8_snu114_brr2_5gan/1'>tri-snRNP</scene> which is the last remaining complex necessary to form the complete spliceosome and is therefore vital for its function. <br />
===U5 snRNA===<br />
The U5 snRNA interacts directly with the 5’ exon before the first step of splicing and with the 5’ and 3’ exons following the first step of splicing <ref name="Keefe">PMID:9430647</ref>. These U5–exon interactions are essential for tethering and aligning the exons for ligation during the second step of splicing <ref name="Keefe" />.<br />
===Brr2===<br />
<Structure load='3hib' size='400' frame='true' align='right' caption='Brr2="/><br />
Brr2 is an ATPase required for unwinding U4/U6 base-pairing before the first step of splicing and for unwinding U2/U6 base-pairing after the second step of splicing <ref>PMID:20659012</ref>. These conformational changes catalyzed by Brr2 are essential for the progression of the spliceosome cycle. <br />
===Prp8===<br />
<Structure load='3e66' size='400' frame='true' align='right' caption='Prp8'/><br />
Prp8 is believed to be the master regulator of the splicing cycle by regulating the spliceosomal DExD/H-box RNA dependent ATPases, specifically Brr2 <ref>PMID:11017191</ref>. Consistent with this regulatory role it has been shown that the C-terminus of Prp8 activates Brr2 helicase function and inhibits Brr20 s U4/U6-dependent ATPase activity in vitro <ref>PMID:19098916</ref>. Structural studies of the Prp8 C-terminus have identified an RNase H-like domain within Prp8 and it has been proposed that this RNase H-domain may form the active site of the spliceosome <ref name="Irish" />.<br />
===Snu114===<br />
<scene name='79/798410/Snu114/1'>Snu114</scene> is the only GTPase associated with the spliceosome and it has multiple contacts with Prp8 as well as U5 snRNA and Brr2 but its role in spliceosome function is not yet well-defined. <ref name="Irish" />.<br />
==Structural Highlights==<br />
Four sequences are highly conserved within the U5 snRNA and it is believed that these sequences play a primary role in the proteins binding capabilities. The four regions are the Stem-loop 1, Internal loop 1, Stem-closing internal loop 1, and Sm protein binding site <ref name="Frank">PMID:8114748</ref>. Stem-loop 1 spans nucleotides 84 to 110 (S. cerevisiae) and consists of a highly conserved 11 nucleotide block with complementary sequences on either side <ref name="Frank" />. These complementary sequences enable local base-pairing to form the stem of the loop <ref name="Frank" />. The U5 snRNA stem-loop 1 is necessary for tethering and aligning exons for ligation during the second step of splicing. The U5 snRNA stem 1 and internal loop 1 (IL1) are also highly conserved between yeast and humans, both in size and in structure <ref name="Frank" />. The U5 snRNA IL1 was found to be the most important region for the association of Prp8, Snu114 and Brr2 with the U5 snRNA <ref>PMID:23857713</ref>. Genetic analysis also identified the U5 snRNA IL1 as being important for Brr2 function <ref name="Frank" />. Overall, the U5 snRNA IL1 appears to be critical for the association of the U5 snRNP proteins Prp8, Snu114 and Brr2 with the U5 snRNA to form the <scene name='79/798410/Prp8_snu114_brr2_5gan/2'>U5 snRNP</scene> <ref name="Frank" />. <br />
===Brr2 Structural Highlights===<br />
<Structure load='3hib' size='400' frame='true' align='right' caption='Brr2'/><br />
Recent structural analysis of Brr2 has revealed that parts of the Sec63 like cassettes of the Brr2 helicase domains resembles the DNA helicase Hel308, hinting at an RNA unwinding action of Brr2 similar to that of the DNA unwinding by helicase Hel308 <ref name="Irish" />. The activity of Brr2 is regulated by the guanine nucleotide state of Snu114 <ref name="Irish" />.<br />
===Prp8 Structural Highlights===<br />
<Structure load='3e66' size='400' frame='true' align='right' caption='Prp8'/><br />
Prp8 is a 280 kDa protein component of the U5 snRNP and is also part of the U4/U6.U5 tri-snRNP <ref name="Irish" />. Prp8 forms a complex with the assembly factor Aar2 and Snu114 without the U5 snRNA in the cytoplasm, however, Aar2 is replaced with Brr2 in the nucleus <ref name="Irish" />.<br />
==Protein Interactions==<br />
The U5 snRNP is one of the five subunits which come together to form the spliceosome and is, therefore, a key protein for eukaryotic mRNA processing. U5 snRNP is a complex which consists of U5 snRNA, Brr2, Snu114, and Prp8. Prp8 also interacts with other proteins of the spliceosome, the snRNAs and extensively with the pre-mRNA <ref name="Irish" />. It has been shown that Prp8 crosslinks directly to the pre-mRNA 5’ splice site, the branch site, and the 3’ splice site as well as U5 and U6 snRNAs localizing it to the heart of the spliceosome <ref name="Irish" />. In yeast, Prp8 makes extensive direct contacts with the U5 snRNA including the highly conserved U5 loop 1 and IL1 <ref name="Irish" />.<br />
Following successful assembly of the U5 snRNP, a <scene name='79/798410/Prp8_snu114_brr2_5gan/1'>tri-snRNP</scene> consisting of U4, U5 and U6 is formed which is then incorporated into the active spliceosome. <Structure load='5gan' size='400' frame='true' align='right' caption='U4/U6.U5 tri-snRNP'/><br />
<br />
<br />
</StructureSection><br />
== References ==<br />
<references/></div>Wally Novakhttps://proteopedia.org/index.php?title=User:Wally_Novak/Sandbox_Whitaker&diff=2955050User:Wally Novak/Sandbox Whitaker2018-10-09T03:03:28Z<p>Wally Novak: </p>
<hr />
<div>==U5 snRNP==<br />
<StructureSection load='1stp' size='340' side='right' caption='Human Spliceosomal Protein U5-15kD'><br />
==Function==<br />
===General Overview===<br />
The U5 snRNP consists of several proteins which are highly conserved from yeast to humans. These proteins consist of <scene name='79/798410/Yeast_prp8/1'>Prp8</scene>, Snu114, <scene name='79/798410/Yeast_brr2/1'>Brr2</scene>, Prp28, Snu40/52K, and <scene name='79/798410/Dib1/1'>Dib1</scene><ref>PMID:11720284</ref>, however, only Prp8, Snu114, and Brr2 remain in complex with the U5 snRNA while the spliceosome is activated <ref name="Irish">PMID:23354046</ref>. These proteins are necessary for the remodeling of the spliceosome and may play a role in maintaining catalytic activity at the active center of the spliceosome <ref name="Irish" />. Assembly of the U5 snRNP is followed by the formation of the U4/U6.U5 <scene name='79/798410/Prp8_snu114_brr2_5gan/1'>tri-snRNP</scene> which is the last remaining complex necessary to form the complete spliceosome and is therefore vital for its function. <br />
===U5 snRNA===<br />
The U5 snRNA interacts directly with the 5’ exon before the first step of splicing and with the 5’ and 3’ exons following the first step of splicing <ref name="Keefe">PMID:9430647</ref>. These U5–exon interactions are essential for tethering and aligning the exons for ligation during the second step of splicing <ref name="Keefe" />.<br />
===Brr2===<br />
<Structure load='3hib' size='400' frame='true' align='right' caption='Brr2="/><br />
Brr2 is an ATPase required for unwinding U4/U6 base-pairing before the first step of splicing and for unwinding U2/U6 base-pairing after the second step of splicing <ref>PMID:20659012</ref>. These conformational changes catalyzed by Brr2 are essential for the progression of the spliceosome cycle. <br />
===Prp8===<br />
<Structure load='3e66' size='400' frame='true' align='right' caption='Prp8'/><br />
Prp8 is believed to be the master regulator of the splicing cycle by regulating the spliceosomal DExD/H-box RNA dependent ATPases, specifically Brr2 <ref>PMID:11017191</ref>. Consistent with this regulatory role it has been shown that the C-terminus of Prp8 activates Brr2 helicase function and inhibits Brr20 s U4/U6-dependent ATPase activity in vitro <ref>PMID:19098916</ref>. Structural studies of the Prp8 C-terminus have identified an RNase H-like domain within Prp8 and it has been proposed that this RNase H-domain may form the active site of the spliceosome <ref name="Irish" />.<br />
===Snu114===<br />
Snu114 is the only GTPase associated with the spliceosome and it has multiple contacts with Prp8 as well as U5 snRNA and Brr2 but its role in spliceosome function is not yet well-defined. <ref name="Irish" />.<br />
==Structural Highlights==<br />
Four sequences are highly conserved within the U5 snRNA and it is believed that these sequences play a primary role in the proteins binding capabilities. The four regions are the Stem-loop 1, Internal loop 1, Stem-closing internal loop 1, and Sm protein binding site <ref name="Frank">PMID:8114748</ref>. Stem-loop 1 spans nucleotides 84 to 110 (S. cerevisiae) and consists of a highly conserved 11 nucleotide block with complementary sequences on either side <ref name="Frank" />. These complementary sequences enable local base-pairing to form the stem of the loop <ref name="Frank" />. The U5 snRNA stem-loop 1 is necessary for tethering and aligning exons for ligation during the second step of splicing. The U5 snRNA stem 1 and internal loop 1 (IL1) are also highly conserved between yeast and humans, both in size and in structure <ref name="Frank" />. The U5 snRNA IL1 was found to be the most important region for the association of Prp8, Snu114 and Brr2 with the U5 snRNA <ref>PMID:23857713</ref>. Genetic analysis also identified the U5 snRNA IL1 as being important for Brr2 function <ref name="Frank" />. Overall, the U5 snRNA IL1 appears to be critical for the association of the U5 snRNP proteins Prp8, Snu114 and Brr2 with the U5 snRNA to form the <scene name='79/798410/Prp8_snu114_brr2_5gan/2'>U5 snRNP</scene> <ref name="Frank" />. <br />
===Brr2 Structural Highlights===<br />
<Structure load='3hib' size='400' frame='true' align='right' caption='Brr2'/><br />
Recent structural analysis of Brr2 has revealed that parts of the Sec63 like cassettes of the Brr2 helicase domains resembles the DNA helicase Hel308, hinting at an RNA unwinding action of Brr2 similar to that of the DNA unwinding by helicase Hel308 <ref name="Irish" />. The activity of Brr2 is regulated by the guanine nucleotide state of Snu114 <ref name="Irish" />.<br />
===Prp8 Structural Highlights===<br />
<Structure load='3e66' size='400' frame='true' align='right' caption='Prp8'/><br />
Prp8 is a 280 kDa protein component of the U5 snRNP and is also part of the U4/U6.U5 tri-snRNP <ref name="Irish" />. Prp8 forms a complex with the assembly factor Aar2 and Snu114 without the U5 snRNA in the cytoplasm, however, Aar2 is replaced with Brr2 in the nucleus <ref name="Irish" />.<br />
==Protein Interactions==<br />
The U5 snRNP is one of the five subunits which come together to form the spliceosome and is, therefore, a key protein for eukaryotic mRNA processing. U5 snRNP is a complex which consists of U5 snRNA, Brr2, Snu114, and Prp8. Prp8 also interacts with other proteins of the spliceosome, the snRNAs and extensively with the pre-mRNA <ref name="Irish" />. It has been shown that Prp8 crosslinks directly to the pre-mRNA 5’ splice site, the branch site, and the 3’ splice site as well as U5 and U6 snRNAs localizing it to the heart of the spliceosome <ref name="Irish" />. In yeast, Prp8 makes extensive direct contacts with the U5 snRNA including the highly conserved U5 loop 1 and IL1 <ref name="Irish" />.<br />
Following successful assembly of the U5 snRNP, a <scene name='79/798410/Prp8_snu114_brr2_5gan/1'>tri-snRNP</scene> consisting of U4, U5 and U6 is formed which is then incorporated into the active spliceosome. <Structure load='5gan' size='400' frame='true' align='right' caption='U4/U6.U5 tri-snRNP'/><br />
<br />
<br />
</StructureSection><br />
== References ==<br />
<references/></div>Wally Novakhttps://proteopedia.org/index.php?title=User:Wally_Novak/Sandbox_Whitaker&diff=2955049User:Wally Novak/Sandbox Whitaker2018-10-09T02:56:49Z<p>Wally Novak: </p>
<hr />
<div>==U5 snRNP==<br />
<StructureSection load='1stp' size='340' side='right' caption='Human Spliceosomal Protein U5-15kD'><br />
==Function==<br />
===General Overview===<br />
The U5 snRNP consists of several proteins which are highly conserved from yeast to humans. These proteins consist of Prp8, Snu114, Brr2, Prp28, Snu40/52K, and <scene name='79/798410/Dib1/1'>Dib1</scene><ref>PMID:11720284</ref>, however, only Prp8, Snu114, and Brr2 remain in complex with the U5 snRNA while the spliceosome is activated <ref name="Irish">PMID:23354046</ref>. These proteins are necessary for the remodeling of the spliceosome and may play a role in maintaining catalytic activity at the active center of the spliceosome <ref name="Irish" />. Assembly of the U5 snRNP is followed by the formation of the U4/U6.U5 tri-snRNP which is the last remaining complex necessary to form the complete spliceosome and is therefore vital for its function. <br />
===U5 snRNA===<br />
The U5 snRNA interacts directly with the 5’ exon before the first step of splicing and with the 5’ and 3’ exons following the first step of splicing <ref name="Keefe">PMID:9430647</ref>. These U5–exon interactions are essential for tethering and aligning the exons for ligation during the second step of splicing <ref name="Keefe" />.<br />
===Brr2===<br />
<Structure load='3hib' size='400' frame='true' align='right' caption='Brr2="/><br />
Brr2 is an ATPase required for unwinding U4/U6 base-pairing before the first step of splicing and for unwinding U2/U6 base-pairing after the second step of splicing <ref>PMID:20659012</ref>. These conformational changes catalyzed by Brr2 are essential for the progression of the spliceosome cycle. <br />
===Prp8===<br />
<Structure load='3e66' size='400' frame='true' align='right' caption='Prp8'/><br />
Prp8 is believed to be the master regulator of the splicing cycle by regulating the spliceosomal DExD/H-box RNA dependent ATPases, specifically Brr2 <ref>PMID:11017191</ref>. Consistent with this regulatory role it has been shown that the C-terminus of Prp8 activates Brr2 helicase function and inhibits Brr20 s U4/U6-dependent ATPase activity in vitro <ref>PMID:19098916</ref>. Structural studies of the Prp8 C-terminus have identified an RNase H-like domain within Prp8 and it has been proposed that this RNase H-domain may form the active site of the spliceosome <ref name="Irish" />.<br />
===Snu114===<br />
Snu114 is the only GTPase associated with the spliceosome and it has multiple contacts with Prp8 as well as U5 snRNA and Brr2 but its role in spliceosome function is not yet well-defined. <ref name="Irish" />.<br />
==Structural Highlights==<br />
Four sequences are highly conserved within the U5 snRNA and it is believed that these sequences play a primary role in the proteins binding capabilities. The four regions are the Stem-loop 1, Internal loop 1, Stem-closing internal loop 1, and Sm protein binding site <ref name="Frank">PMID:8114748</ref>. Stem-loop 1 spans nucleotides 84 to 110 (S. cerevisiae) and consists of a highly conserved 11 nucleotide block with complementary sequences on either side <ref name="Frank" />. These complementary sequences enable local base-pairing to form the stem of the loop <ref name="Frank" />. The U5 snRNA stem-loop 1 is necessary for tethering and aligning exons for ligation during the second step of splicing. The U5 snRNA stem 1 and internal loop 1 (IL1) are also highly conserved between yeast and humans, both in size and in structure <ref name="Frank" />. The U5 snRNA IL1 was found to be the most important region for the association of Prp8, Snu114 and Brr2 with the U5 snRNA <ref>PMID:23857713</ref>. Genetic analysis also identified the U5 snRNA IL1 as being important for Brr2 function <ref name="Frank" />. Overall, the U5 snRNA IL1 appears to be critical for the association of the U5 snRNP proteins Prp8, Snu114 and Brr2 with the U5 snRNA to form the <scene name='79/798410/Prp8_snu114_brr2_5gan/2'>U5 snRNP</scene> <ref name="Frank" />. <br />
===Brr2 Structural Highlights===<br />
<Structure load='3hib' size='400' frame='true' align='right' caption='Brr2'/><br />
Recent structural analysis of Brr2 has revealed that parts of the Sec63 like cassettes of the Brr2 helicase domains resembles the DNA helicase Hel308, hinting at an RNA unwinding action of Brr2 similar to that of the DNA unwinding by helicase Hel308 <ref name="Irish" />. The activity of Brr2 is regulated by the guanine nucleotide state of Snu114 <ref name="Irish" />.<br />
===Prp8 Structural Highlights===<br />
<Structure load='3e66' size='400' frame='true' align='right' caption='Prp8'/><br />
Prp8 is a 280 kDa protein component of the U5 snRNP and is also part of the U4/U6.U5 tri-snRNP <ref name="Irish" />. Prp8 forms a complex with the assembly factor Aar2 and Snu114 without the U5 snRNA in the cytoplasm, however, Aar2 is replaced with Brr2 in the nucleus <ref name="Irish" />.<br />
==Protein Interactions==<br />
The U5 snRNP is one of the five subunits which come together to form the spliceosome and is, therefore, a key protein for eukaryotic mRNA processing. U5 snRNP is a complex which consists of U5 snRNA, Brr2, Snu114, and Prp8. Prp8 also interacts with other proteins of the spliceosome, the snRNAs and extensively with the pre-mRNA <ref name="Irish" />. It has been shown that Prp8 crosslinks directly to the pre-mRNA 5’ splice site, the branch site, and the 3’ splice site as well as U5 and U6 snRNAs localizing it to the heart of the spliceosome <ref name="Irish" />. In yeast, Prp8 makes extensive direct contacts with the U5 snRNA including the highly conserved U5 loop 1 and IL1 <ref name="Irish" />.<br />
Following successful assembly of the U5 snRNP, a tri-snRNP consisting of U4, U5 and U6 is formed which is then incorporated into the active spliceosome. <Structure load='5gan' size='400' frame='true' align='right' caption='U4/U6.U5 tri-snRNP'/><br />
<br />
<br />
</StructureSection><br />
== References ==<br />
<references/></div>Wally Novakhttps://proteopedia.org/index.php?title=Rde_4_sandbox&diff=2955048Rde 4 sandbox2018-10-09T02:54:12Z<p>Wally Novak: </p>
<hr />
<div>==Introduction==<br />
<br />
RDE-4 was identified through a genome-wide screening as a gene responsible for greatly reducing or abolishing RNAi in C. elegans. In organisms ranging from Arabidopsis to humans, Dicer requires dsRNA-binding proteins (dsRBPs) to carry out its roles in RNA interference (RNAi) and micro-RNA (miRNA) processing. In Caenorhabditis elegans, the dsRBP RDE-4 acts with Dicer during the initiation of RNAi, when long dsRNA is cleaved to small interfering RNAs (siRNAs). RDE-4 is not sequence-specific, however, RDE-4 binds with higher affinity to long dsRNA. Interestingly, RDE- 4 is the only protein that exhibits micromolar affinity for dsRNA in the absence of co-operativity. In addition, RDE-4 is a homodimer in solution. <ref name=first>DOI: 10.1261/rna.2338706</ref><br />
<br />
<Structure load='2LTS' size='350' frame='true' align='right' caption='Structure of RDE-4 (pdb code 2LTS)'/><br />
<br />
<Structure load='2LTR' size='350' frame='true' align='right' caption='Structure of RDE-4 (pdb code 2LTR)'/><br />
<br />
== Structure of RDE-4 ==<br />
<br />
RDE-4 has five major regions: an <scene name='79/798389/N-terminal_of_rde-4/1'>N-terminal region (residues 1-43)</scene>, two dsRBDs <scene name='79/798389/Dsrbd_residues_44-108/1'>(residues 44-108)</scene> and <scene name='79/798389/Dsrbd_residues_170-235/2'>(residues 170-235)</scene>, a <scene name='79/798389/Long_linker_of_rde-4/1'>Long Linker (residues 109-169)</scene>, and a C-terminal domain (residues 236–385). Amino acid residues 1–32 and 136–151 adopt a random coiled structure and do not make any contact within itself or with the rest of the structure. Parts of the linker, residues 109–135 and 152–169, assume an α-helix and an extended loop structure, respectively. There is not any long-range nuclear Overhauser effect information between regions 1–135 and 152– 243, suggesting that the unstructured part of the linker 136–151 separates both of these regions. The region encompassed by 236–243 folds into an α-helix. The amino acid regions 44–108 and 170–235 form dsRBD1 and dsRBD2, respectively. The hydrophobic residues Leu45, Val47, Leu48, Val55, Trp62, Met73, Leu75, Leu77, Ile80, Val82, Leu101, and Val105 stabilize dsRBD1, and Val171, Leu174, Leu183, Val201, Met205, Met227, and Leu232 form the core of dsRBD2. Apart from the canonical dsRBD fold, there are additional secondary-structural elements that are observed in both RDE-4D1 and RDE-4D2. <ref name=second>DOI:DOI: 10.1042/BJ20131347</ref> <br />
<br />
== Function ==<br />
<br />
In C. elegans, exogenous dsRNA is detected and bound by RDE-4, which stimulates dicer activity. RDE-4 initiates the siRNA pathway by binding to long dsRNA and assisting Dcr-1, a Dicer1 homologue, to facilitate siRNA production. RDE-4 interacts with Dcr-1, RDE-1, DRH-1 (Dicer-related helicase 1) and long dsRNA, which thereby suggests that RDE-4 is required only for the initiation of the RNAi pathway to generate siRNA, although high levels of dsRNA abrogate RDE-4’s role in the RNAi initiation. RDE-4 co- operatively binds to long dsRNA with nanomolar affinity, but exhibits micromolar affinity for short dsRNA, and the enhanced affinity arises from binding of several RDE-4 molecules to a single long dsRNA. <ref name=third>DOI: 10.1016/j.jmb.2008.10.002</ref> The C-terminal region of RDE-4 is necessary and sufficient to induce homodimerization RDE-4 linker– dsRBD2 are absolutely essential in C. elegans gene silencing and RDE-4 dsRBD1 and the C-terminal domain do not have any primary role in vivo. RDE-4 recognizes the A-form of nucleic acid structure adopted by dsRNA and the interaction is sequence-independent.<ref name=second/> RDE-4 dimerization is important for the assembly of active RDE-4/Dicer complexes via one of two proposed scenarios, proper Dicer recruitment to dsRNA, or facilitating Dicer dimerization. <ref name=first/> <br />
<br />
<br />
== References ==<br />
<references/></div>Wally Novakhttps://proteopedia.org/index.php?title=Rde_4_sandbox&diff=2955047Rde 4 sandbox2018-10-09T02:50:49Z<p>Wally Novak: </p>
<hr />
<div>==Introduction==<br />
<br />
RDE-4 was identified through a genome-wide screening as a gene responsible for greatly reducing or abolishing RNAi in C. elegans. In organisms ranging from Arabidopsis to humans, Dicer requires dsRNA-binding proteins (dsRBPs) to carry out its roles in RNA interference (RNAi) and micro-RNA (miRNA) processing. In Caenorhabditis elegans, the dsRBP RDE-4 acts with Dicer during the initiation of RNAi, when long dsRNA is cleaved to small interfering RNAs (siRNAs). RDE-4 is not sequence-specific, however, RDE-4 binds with higher affinity to long dsRNA. Interestingly, RDE- 4 is the only protein that exhibits micromolar affinity for dsRNA in the absence of co-operativity. In addition, RDE-4 is a homodimer in solution. <ref name=first>DOI: 10.1261/rna.2338706</ref><br />
<br />
<Structure load='2LTS' size='350' frame='true' align='right' caption='Structure of RDE-4 (pdb code 2LTS)'/><br />
<br />
<Structure load='2LTR' size='350' frame='true' align='right' caption='Structure of RDE-4 (pdb code 2LTR)'/><br />
<br />
== Structure of RDE-4 ==<br />
<br />
RDE-4 has five major regions: an <scene name='79/798389/N-terminal_of_rde-4/1'>N-terminal region (residues 1-43)</scene>, two dsRBDs <scene name='79/798389/Dsrbd_residues_44-108/1'>(residues 44-108)</scene> and <scene name='79/798389/Dsrbd_residues_170-235/2'>(residues 170-235)</scene>, a <scene name='79/798389/Long_linker_of_rde-4/1'>Long Linker (residues 109-169)</scene>, and a C-terminal domain (residues 236–385). Amino acid residues 1–32 and 136–151 adopt a random coiled structure and do not make any contact within itself or with the rest of the structure. Parts of the linker, residues 109–135 and 152–169, assume an α-helix and an extended loop structure, respectively. There is not any long-range nuclear Overhauser effect information between regions 1–135 and 152– 243, suggesting that the unstructured part of the linker 136–151 separates both of these regions. The region encompassed by 236–243 folds into an α-helix. The amino acid regions 44–108 and 170–235 form dsRBD1 and dsRBD2, respectively. The hydrophobic residues Leu45, Val47, Leu48, Val55, Trp62, Met73, Leu75, Leu77, Ile80, Val82, Leu101, and Val105 stabilize dsRBD1, and Val171, Leu174, Leu183, Val201, Met205, Met227, and Leu232 form the core of dsRBD2. Apart from the canonical dsRBD fold, there are additional secondary-structural elements that are observed in both RDE-4D1 and RDE-4D2. <ref name=second>DOI:DOI: 10.1042/BJ20131347</ref> <br />
<br />
== Function ==<br />
<br />
In C. elegans, exogenous dsRNA is detected and bound by RDE-4, which stimulates dicer activity. RDE-4 initiates the siRNA pathway by binding to long dsRNA and assisting Dcr-1, a Dicer1 homologue, to facilitate siRNA production. RDE-4 interacts with Dcr-1, RDE-1, DRH-1 (Dicer-related helicase 1) and long dsRNA, which thereby suggests that RDE-4 is required only for the initiation of the RNAi pathway to generate siRNA, although high levels of dsRNA abrogate RDE-4’s role in the RNAi initiation. RDE-4 co- operatively binds to long dsRNA with nanomolar affinity, but exhibits micromolar affinity for short dsRNA, and the enhanced affinity arises from binding of several RDE-4 molecules to a single long dsRNA. <ref name=third>DOI: 10.1016/j.jmb.2008.10.002</ref> The C-terminal region of RDE-4 is necessary and sufficient to induce homodimerization RDE-4 linker– dsRBD2 are absolutely essential in C. elegans gene silencing and RDE-4 dsRBD1 and the C-terminal domain do not have any primary role in vivo. RDE-4 recognizes the A-form of nucleic acid structure adopted by dsRNA and the interaction is sequence-independent.<ref name=second/>(Purple citation) RDE-4 dimerization is important for the assembly of active RDE-4/Dicer complexes via one of two proposed scenarios, proper Dicer recruitment to dsRNA, or facilitating Dicer dimerization. <ref name=first/> <br />
<br />
<br />
== References ==<br />
<references/></div>Wally Novakhttps://proteopedia.org/index.php?title=User:Wally_Novak/Sandbox_Whitaker&diff=2955046User:Wally Novak/Sandbox Whitaker2018-10-09T02:50:44Z<p>Wally Novak: </p>
<hr />
<div>==U5 snRNP==<br />
<StructureSection load='1stp' size='340' side='right' caption='Human Spliceosomal Protein U5-15kD'><br />
==Function==<br />
===General Overview===<br />
The U5 snRNP consists of several proteins which are highly conserved from yeast to humans. These proteins consist of Prp8, Snu114, Brr2, Prp28, Snu40/52K, and Dib1<ref>PMID:11720284</ref>, however, only Prp8, Snu114, and Brr2 remain in complex with the U5 snRNA while the spliceosome is activated <ref name="Irish">PMID:23354046</ref>. These proteins are necessary for the remodeling of the spliceosome and may play a role in maintaining catalytic activity at the active center of the spliceosome <ref name="Irish" />. Assembly of the U5 snRNP is followed by the formation of the U4/U6.U5 tri-snRNP which is the last remaining complex necessary to form the complete spliceosome and is therefore vital for its function. <br />
===U5 snRNA===<br />
The U5 snRNA interacts directly with the 5’ exon before the first step of splicing and with the 5’ and 3’ exons following the first step of splicing <ref name="Keefe">PMID:9430647</ref>. These U5–exon interactions are essential for tethering and aligning the exons for ligation during the second step of splicing <ref name="Keefe" />.<br />
===Brr2===<br />
<Structure load='3hib' size='400' frame='true' align='right' caption='Brr2="/><br />
Brr2 is an ATPase required for unwinding U4/U6 base-pairing before the first step of splicing and for unwinding U2/U6 base-pairing after the second step of splicing <ref>PMID:20659012</ref>. These conformational changes catalyzed by Brr2 are essential for the progression of the spliceosome cycle. <br />
===Prp8===<br />
<Structure load='3e66' size='400' frame='true' align='right' caption='Prp8'/><br />
Prp8 is believed to be the master regulator of the splicing cycle by regulating the spliceosomal DExD/H-box RNA dependent ATPases, specifically Brr2 <ref>PMID:11017191</ref>. Consistent with this regulatory role it has been shown that the C-terminus of Prp8 activates Brr2 helicase function and inhibits Brr20 s U4/U6-dependent ATPase activity in vitro <ref>PMID:19098916</ref>. Structural studies of the Prp8 C-terminus have identified an RNase H-like domain within Prp8 and it has been proposed that this RNase H-domain may form the active site of the spliceosome <ref name="Irish" />.<br />
===Snu114===<br />
Snu114 is the only GTPase associated with the spliceosome and it has multiple contacts with Prp8 as well as U5 snRNA and Brr2 but its role in spliceosome function is not yet well-defined. <ref name="Irish" />.<br />
==Structural Highlights==<br />
Four sequences are highly conserved within the U5 snRNA and it is believed that these sequences play a primary role in the proteins binding capabilities. The four regions are the Stem-loop 1, Internal loop 1, Stem-closing internal loop 1, and Sm protein binding site <ref name="Frank">PMID:8114748</ref>. Stem-loop 1 spans nucleotides 84 to 110 (S. cerevisiae) and consists of a highly conserved 11 nucleotide block with complementary sequences on either side <ref name="Frank" />. These complementary sequences enable local base-pairing to form the stem of the loop <ref name="Frank" />. The U5 snRNA stem-loop 1 is necessary for tethering and aligning exons for ligation during the second step of splicing. The U5 snRNA stem 1 and internal loop 1 (IL1) are also highly conserved between yeast and humans, both in size and in structure <ref name="Frank" />. The U5 snRNA IL1 was found to be the most important region for the association of Prp8, Snu114 and Brr2 with the U5 snRNA <ref>PMID:23857713</ref>. Genetic analysis also identified the U5 snRNA IL1 as being important for Brr2 function <ref name="Frank" />. Overall, the U5 snRNA IL1 appears to be critical for the association of the U5 snRNP proteins Prp8, Snu114 and Brr2 with the U5 snRNA to form the U5 snRNP <ref name="Frank" />. <br />
===Brr2 Structural Highlights===<br />
<Structure load='3hib' size='400' frame='true' align='right' caption='Brr2'/><br />
Recent structural analysis of Brr2 has revealed that parts of the Sec63 like cassettes of the Brr2 helicase domains resembles the DNA helicase Hel308, hinting at an RNA unwinding action of Brr2 similar to that of the DNA unwinding by helicase Hel308 <ref name="Irish" />. The activity of Brr2 is regulated by the guanine nucleotide state of Snu114 <ref name="Irish" />.<br />
===Prp8 Structural Highlights===<br />
<Structure load='3e66' size='400' frame='true' align='right' caption='Prp8'/><br />
Prp8 is a 280 kDa protein component of the U5 snRNP and is also part of the U4/U6.U5 tri-snRNP <ref name="Irish" />. Prp8 forms a complex with the assembly factor Aar2 and Snu114 without the U5 snRNA in the cytoplasm, however, Aar2 is replaced with Brr2 in the nucleus <ref name="Irish" />.<br />
==Protein Interactions==<br />
The U5 snRNP is one of the five subunits which come together to form the spliceosome and is, therefore, a key protein for eukaryotic mRNA processing. U5 snRNP is a complex which consists of U5 snRNA, Brr2, Snu114, and Prp8. Prp8 also interacts with other proteins of the spliceosome, the snRNAs and extensively with the pre-mRNA <ref name="Irish" />. It has been shown that Prp8 crosslinks directly to the pre-mRNA 5’ splice site, the branch site, and the 3’ splice site as well as U5 and U6 snRNAs localizing it to the heart of the spliceosome <ref name="Irish" />. In yeast, Prp8 makes extensive direct contacts with the U5 snRNA including the highly conserved U5 loop 1 and IL1 <ref name="Irish" />.<br />
Following successful assembly of the U5 snRNP, a tri-snRNP consisting of U4, U5 and U6 is formed which is then incorporated into the active spliceosome. <Structure load='5gan' size='400' frame='true' align='right' caption='U4/U6.U5 tri-snRNP'/><br />
<br />
<br />
</StructureSection><br />
== References ==<br />
<references/></div>Wally Novakhttps://proteopedia.org/index.php?title=Rde_4_sandbox&diff=2955045Rde 4 sandbox2018-10-09T02:49:51Z<p>Wally Novak: </p>
<hr />
<div>==Introduction==<br />
<br />
RDE-4 was identified through a genome-wide screening as a gene responsible for greatly reducing or abolishing RNAi in C. elegans. In organisms ranging from Arabidopsis to humans, Dicer requires dsRNA-binding proteins (dsRBPs) to carry out its roles in RNA interference (RNAi) and micro-RNA (miRNA) processing. In Caenorhabditis elegans, the dsRBP RDE-4 acts with Dicer during the initiation of RNAi, when long dsRNA is cleaved to small interfering RNAs (siRNAs). RDE-4 is not sequence-specific, however, RDE-4 binds with higher affinity to long dsRNA. Interestingly, RDE- 4 is the only protein that exhibits micromolar affinity for dsRNA in the absence of co-operativity. In addition, RDE-4 is a homodimer in solution. <ref name=first>DOI: 10.1261/rna.2338706</ref><br />
<br />
<Structure load='2LTS' size='350' frame='true' align='right' caption='Structure of RDE-4 (pdb code 2LTS)'/><br />
<br />
<Structure load='2LTR' size='350' frame='true' align='right' caption='Structure of RDE-4 (pdb code 2LTR)'/><br />
<br />
== Structure of RDE-4 ==<br />
<br />
RDE-4 has five major regions: an <scene name='79/798389/N-terminal_of_rde-4/1'>N-terminal region (residues 1-43)</scene>, two dsRBDs <scene name='79/798389/Dsrbd_residues_44-108/1'>(residues 44-108)</scene> and <scene name='79/798389/Dsrbd_residues_170-235/2'>(residues 170-235)</scene> (residues 44–108 and 170–235), a <scene name='79/798389/Long_linker_of_rde-4/1'>Long Linker (residues 109-169)</scene>, and a C-terminal domain (residues 236–385). Amino acid residues 1–32 and 136–151 adopt a random coiled structure and do not make any contact within itself or with the rest of the structure. Parts of the linker, residues 109–135 and 152–169, assume an α-helix and an extended loop structure, respectively. There is not any long-range nuclear Overhauser effect information between regions 1–135 and 152– 243, suggesting that the unstructured part of the linker 136–151 separates both of these regions. The region encompassed by 236–243 folds into an α-helix. The amino acid regions 44–108 and 170–235 form dsRBD1 and dsRBD2, respectively. The hydrophobic residues Leu45, Val47, Leu48, Val55, Trp62, Met73, Leu75, Leu77, Ile80, Val82, Leu101, and Val105 stabilize dsRBD1, and Val171, Leu174, Leu183, Val201, Met205, Met227, and Leu232 form the core of dsRBD2. Apart from the canonical dsRBD fold, there are additional secondary-structural elements that are observed in both RDE-4D1 and RDE-4D2. <ref name=second>DOI:DOI: 10.1042/BJ20131347</ref> <br />
<br />
== Function ==<br />
<br />
In C. elegans, exogenous dsRNA is detected and bound by RDE-4, which stimulates dicer activity. RDE-4 initiates the siRNA pathway by binding to long dsRNA and assisting Dcr-1, a Dicer1 homologue, to facilitate siRNA production. RDE-4 interacts with Dcr-1, RDE-1, DRH-1 (Dicer-related helicase 1) and long dsRNA, which thereby suggests that RDE-4 is required only for the initiation of the RNAi pathway to generate siRNA, although high levels of dsRNA abrogate RDE-4’s role in the RNAi initiation. RDE-4 co- operatively binds to long dsRNA with nanomolar affinity, but exhibits micromolar affinity for short dsRNA, and the enhanced affinity arises from binding of several RDE-4 molecules to a single long dsRNA. <ref name=third>DOI: 10.1016/j.jmb.2008.10.002</ref> The C-terminal region of RDE-4 is necessary and sufficient to induce homodimerization RDE-4 linker– dsRBD2 are absolutely essential in C. elegans gene silencing and RDE-4 dsRBD1 and the C-terminal domain do not have any primary role in vivo. RDE-4 recognizes the A-form of nucleic acid structure adopted by dsRNA and the interaction is sequence-independent.<ref name=second/>(Purple citation) RDE-4 dimerization is important for the assembly of active RDE-4/Dicer complexes via one of two proposed scenarios, proper Dicer recruitment to dsRNA, or facilitating Dicer dimerization. <ref name=first/> <br />
<br />
<br />
== References ==<br />
<references/></div>Wally Novakhttps://proteopedia.org/index.php?title=Rde_4_sandbox&diff=2955044Rde 4 sandbox2018-10-09T02:46:25Z<p>Wally Novak: </p>
<hr />
<div>==Introduction==<br />
<br />
RDE-4 was identified through a genome-wide screening as a gene responsible for greatly reducing or abolishing RNAi in C. elegans. In organisms ranging from Arabidopsis to humans, Dicer requires dsRNA-binding proteins (dsRBPs) to carry out its roles in RNA interference (RNAi) and micro-RNA (miRNA) processing. In Caenorhabditis elegans, the dsRBP RDE-4 acts with Dicer during the initiation of RNAi, when long dsRNA is cleaved to small interfering RNAs (siRNAs). RDE-4 is not sequence-specific, however, RDE-4 binds with higher affinity to long dsRNA. Interestingly, RDE- 4 is the only protein that exhibits micromolar affinity for dsRNA in the absence of co-operativity. In addition, RDE-4 is a homodimer in solution. <ref name=first>DOI: 10.1261/rna.2338706</ref><br />
<br />
<Structure load='2LTS' size='350' frame='true' align='right' caption='Structure of RDE-4 (pdb code 2LTS)'/><br />
<br />
<Structure load='2LTR' size='350' frame='true' align='right' caption='Structure of RDE-4 (pdb code 2LTR)'/><br />
<br />
== Structure of RDE-4 ==<br />
<br />
RDE-4 has five major regions: an <scene name='79/798389/N-terminal_of_rde-4/1'>N-terminal region (residues 1-43)</scene>, two dsRBDs <scene name='79/798389/Dsrbd_residues_44-108/1'>(residues 44-108)</scene> and <scene name='79/798389/Dsrbd_residues_170-235/2'>(residues 170-235)</scene> (residues 44–108 and 170–235), a <scene name='79/798389/Long_linker_of_rde-4/1'>Long Linker (residues 109-169)</scene>, and a C-terminal domain (residues 236–385). Amino acid residues 1–32 and 136–151 adopt a random coiled structure and do not make any contact within itself or with the rest of the structure. Parts of the linker, residues 109–135 and 152–169, assume an α-helix and an extended loop structure, respectively. There is not any long-range nuclear Overhauser effect information between regions 1–135 and 152– 243, suggesting that the unstructured part of the linker 136–151 separates both of these regions. The region encompassed by 236–243 folds into an α-helix. The amino acid regions 44–108 and 170–235 form dsRBD1 and dsRBD2, respectively. The hydrophobic residues Leu45, Val47, Leu48, Val55, Trp62, Met73, Leu75, Leu77, Ile80, Val82, Leu101, and Val105 stabilize dsRBD1, and Val171, Leu174, Leu183, Val201, Met205, Met227, and Leu232 form the core of dsRBD2. Apart from the canonical dsRBD fold, there are additional secondary-structural elements that are observed in both RDE-4D1 and RDE-4D2. <ref name=second>DOI:DOI: 10.1042/BJ20131347</ref> <br />
<br />
== Function ==<br />
<br />
In C. elegans, exogenous dsRNA is detected and bound by RDE-4, which stimulates dicer activity. RDE-4 initiates the siRNA pathway by binding to long dsRNA and assisting Dcr-1, a Dicer1 homologue, to facilitate siRNA production. RDE-4 interacts with Dcr-1, RDE-1, DRH-1 (Dicer-related helicase 1) and long dsRNA, which thereby suggests that RDE-4 is required only for the initiation of the RNAi pathway to generate siRNA, although high levels of dsRNA abrogate RDE-4’s role in the RNAi initiation. RDE-4 co- operatively binds to long dsRNA with nanomolar affinity, but exhibits micromolar affinity for short dsRNA, and the enhanced affinity arises from binding of several RDE-4 molecules to a single long dsRNA. <ref name=third>DOI: 10.1016/j.jmb.2008.10.002</ref> The C-terminal region of RDE-4 is necessary and sufficient to induce homodimerization RDE-4 linker– dsRBD2 are absolutely essential in C. elegans gene silencing and RDE-4 dsRBD1 and the C-terminal domain do not have any primary role in vivo. RDE-4 recognizes the A-form of nucleic acid structure adopted by dsRNA and the interaction is sequence-independent.<ref name=second/>(Purple citation) RDE-4 dimerization is important for the assembly of active RDE-4/Dicer complexes via one of two proposed scenarios, proper Dicer recruitment to dsRNA, or facilitating Dicer dimerization. <ref name=first/> <br />
<br />
<br />
</StructureSection><br />
== References ==<br />
<references/></div>Wally Novakhttps://proteopedia.org/index.php?title=Rde_4_sandbox&diff=2955043Rde 4 sandbox2018-10-09T02:45:33Z<p>Wally Novak: </p>
<hr />
<div>==Introduction==<br />
<br />
RDE-4 was identified through a genome-wide screening as a gene responsible for greatly reducing or abolishing RNAi in C. elegans. In organisms ranging from Arabidopsis to humans, Dicer requires dsRNA-binding proteins (dsRBPs) to carry out its roles in RNA interference (RNAi) and micro-RNA (miRNA) processing. In Caenorhabditis elegans, the dsRBP RDE-4 acts with Dicer during the initiation of RNAi, when long dsRNA is cleaved to small interfering RNAs (siRNAs). RDE-4 is not sequence-specific, however, RDE-4 binds with higher affinity to long dsRNA. Interestingly, RDE- 4 is the only protein that exhibits micromolar affinity for dsRNA in the absence of co-operativity. In addition, RDE-4 is a homodimer in solution. <ref name=first>DOI: 10.1261/rna.2338706</ref> (Yellow Citation)<br />
<br />
<Structure load='2LTS' size='350' frame='true' align='right' caption='Structure of RDE-4 (pdb code 2LTS)'/><br />
<br />
<Structure load='2LTR' size='350' frame='true' align='right' caption='Structure of RDE-4 (pdb code 2LTR)'/><br />
<br />
== Structure of RDE-4 ==<br />
<br />
RDE-4 has five major regions: an <scene name='79/798389/N-terminal_of_rde-4/1'>N-terminal region (residues 1-43)</scene>, two dsRBDs <scene name='79/798389/Dsrbd_residues_44-108/1'>(residues 44-108)</scene> and <scene name='79/798389/Dsrbd_residues_170-235/2'>(residues 170-235)</scene> (residues 44–108 and 170–235), a <scene name='79/798389/Long_linker_of_rde-4/1'>Long Linker (residues 109-169)</scene>, and a C-terminal domain (residues 236–385). Amino acid residues 1–32 and 136–151 adopt a random coiled structure and do not make any contact within itself or with the rest of the structure. Parts of the linker, residues 109–135 and 152–169, assume an α-helix and an extended loop structure, respectively. There is not any long-range nuclear Overhauser effect information between regions 1–135 and 152– 243, suggesting that the unstructured part of the linker 136–151 separates both of these regions. The region encompassed by 236–243 folds into an α-helix. The amino acid regions 44–108 and 170–235 form dsRBD1 and dsRBD2, respectively. The hydrophobic residues Leu45, Val47, Leu48, Val55, Trp62, Met73, Leu75, Leu77, Ile80, Val82, Leu101, and Val105 stabilize dsRBD1, and Val171, Leu174, Leu183, Val201, Met205, Met227, and Leu232 form the core of dsRBD2. Apart from the canonical dsRBD fold, there are additional secondary-structural elements that are observed in both RDE-4D1 and RDE-4D2. <ref name=second>DOI:DOI: 10.1042/BJ20131347</ref> (Purple citation)<br />
<br />
== Function ==<br />
<br />
In C. elegans, exogenous dsRNA is detected and bound by RDE-4, which stimulates dicer activity. RDE-4 initiates the siRNA pathway by binding to long dsRNA and assisting Dcr-1, a Dicer1 homologue, to facilitate siRNA production. RDE-4 interacts with Dcr-1, RDE-1, DRH-1 (Dicer-related helicase 1) and long dsRNA, which thereby suggests that RDE-4 is required only for the initiation of the RNAi pathway to generate siRNA, although high levels of dsRNA abrogate RDE-4’s role in the RNAi initiation. RDE-4 co- operatively binds to long dsRNA with nanomolar affinity, but exhibits micromolar affinity for short dsRNA, and the enhanced affinity arises from binding of several RDE-4 molecules to a single long dsRNA. <ref name=third>DOI: 10.1016/j.jmb.2008.10.002</ref> (Blue citation) The C-terminal region of RDE-4 is necessary and sufficient to induce homodimerization RDE-4 linker– dsRBD2 are absolutely essential in C. elegans gene silencing and RDE-4 dsRBD1 and the C-terminal domain do not have any primary role in vivo. RDE-4 recognizes the A-form of nucleic acid structure adopted by dsRNA and the interaction is sequence-independent.<ref name=second/>(Purple citation) RDE-4 dimerization is important for the assembly of active RDE-4/Dicer complexes via one of two proposed scenarios, proper Dicer recruitment to dsRNA, or facilitating Dicer dimerization. <ref name=first/> (Yellow citation)<br />
<br />
<br />
This is a sample scene created with SAT to <scene name="/12/3456/Sample/1">color</scene> by Group, and another to make <scene name="/12/3456/Sample/2">a transparent representation</scene> of the protein. You can make your own scenes on SAT starting from scratch or loading and editing one of these sample scenes.<br />
<br />
</StructureSection><br />
== References ==<br />
<references/></div>Wally Novakhttps://proteopedia.org/index.php?title=Rde_4_sandbox&diff=2955042Rde 4 sandbox2018-10-09T02:42:39Z<p>Wally Novak: </p>
<hr />
<div>==Introduction==<br />
<br />
RDE-4 was identified through a genome-wide screening as a gene responsible for greatly reducing or abolishing RNAi in C. elegans. In organisms ranging from Arabidopsis to humans, Dicer requires dsRNA-binding proteins (dsRBPs) to carry out its roles in RNA interference (RNAi) and micro-RNA (miRNA) processing. In Caenorhabditis elegans, the dsRBP RDE-4 acts with Dicer during the initiation of RNAi, when long dsRNA is cleaved to small interfering RNAs (siRNAs). RDE-4 is not sequence-specific, however, RDE-4 binds with higher affinity to long dsRNA. Interestingly, RDE- 4 is the only protein that exhibits micromolar affinity for dsRNA in the absence of co-operativity. In addition, RDE-4 is a homodimer in solution. <ref name=first>DOI: 10.1261/rna.2338706</ref> (Yellow Citation)<br />
<br />
<Structure load='2LTS' size='350' frame='true' align='right' caption='Structure of RDE-4 (pdb code 2LTS)'/><br />
<br />
<Structure load='2LTR' size='350' frame='true' align='right' caption='Structure of RDE-4 (pdb code 2LTR)'/><br />
<br />
== Structure of RDE-4 ==<br />
<br />
RDE-4 has five major regions: an <scene name='79/798389/N-terminal_of_rde-4/1'>N-terminal region (residues 1-43)</scene>, two dsRBDs <scene name='79/798389/Dsrbd_residues_44-108/1'>(residues 44-108)</scene> (residues 44–108 and 170–235), a <scene name='79/798389/Long_linker_of_rde-4/1'>Long Linker (residues 109-169)</scene>, and a C-terminal domain (residues 236–385). Amino acid residues 1–32 and 136–151 adopt a random coiled structure and do not make any contact within itself or with the rest of the structure. Parts of the linker, residues 109–135 and 152–169, assume an α-helix and an extended loop structure, respectively. There is not any long-range nuclear Overhauser effect information between regions 1–135 and 152– 243, suggesting that the unstructured part of the linker 136–151 separates both of these regions. The region encompassed by 236–243 folds into an α-helix. The amino acid regions 44–108 and 170–235 form dsRBD1 and dsRBD2, respectively. The hydrophobic residues Leu45, Val47, Leu48, Val55, Trp62, Met73, Leu75, Leu77, Ile80, Val82, Leu101, and Val105 stabilize dsRBD1, and Val171, Leu174, Leu183, Val201, Met205, Met227, and Leu232 form the core of dsRBD2. Apart from the canonical dsRBD fold, there are additional secondary-structural elements that are observed in both RDE-4D1 and RDE-4D2. <ref name=second>DOI:DOI: 10.1042/BJ20131347</ref> (Purple citation)<br />
<br />
== Function ==<br />
<br />
In C. elegans, exogenous dsRNA is detected and bound by RDE-4, which stimulates dicer activity. RDE-4 initiates the siRNA pathway by binding to long dsRNA and assisting Dcr-1, a Dicer1 homologue, to facilitate siRNA production. RDE-4 interacts with Dcr-1, RDE-1, DRH-1 (Dicer-related helicase 1) and long dsRNA, which thereby suggests that RDE-4 is required only for the initiation of the RNAi pathway to generate siRNA, although high levels of dsRNA abrogate RDE-4’s role in the RNAi initiation. RDE-4 co- operatively binds to long dsRNA with nanomolar affinity, but exhibits micromolar affinity for short dsRNA, and the enhanced affinity arises from binding of several RDE-4 molecules to a single long dsRNA. <ref name=third>DOI: 10.1016/j.jmb.2008.10.002</ref> (Blue citation) The C-terminal region of RDE-4 is necessary and sufficient to induce homodimerization RDE-4 linker– dsRBD2 are absolutely essential in C. elegans gene silencing and RDE-4 dsRBD1 and the C-terminal domain do not have any primary role in vivo. RDE-4 recognizes the A-form of nucleic acid structure adopted by dsRNA and the interaction is sequence-independent.<ref name=second/>(Purple citation) RDE-4 dimerization is important for the assembly of active RDE-4/Dicer complexes via one of two proposed scenarios, proper Dicer recruitment to dsRNA, or facilitating Dicer dimerization. <ref name=first/> (Yellow citation)<br />
<br />
<br />
This is a sample scene created with SAT to <scene name="/12/3456/Sample/1">color</scene> by Group, and another to make <scene name="/12/3456/Sample/2">a transparent representation</scene> of the protein. You can make your own scenes on SAT starting from scratch or loading and editing one of these sample scenes.<br />
<br />
</StructureSection><br />
== References ==<br />
<references/></div>Wally Novakhttps://proteopedia.org/index.php?title=Rde_4_sandbox&diff=2955041Rde 4 sandbox2018-10-09T02:35:02Z<p>Wally Novak: </p>
<hr />
<div>==Introduction==<br />
<br />
RDE-4 was identified through a genome-wide screening as a gene responsible for greatly reducing or abolishing RNAi in C. elegans. In organisms ranging from Arabidopsis to humans, Dicer requires dsRNA-binding proteins (dsRBPs) to carry out its roles in RNA interference (RNAi) and micro-RNA (miRNA) processing. In Caenorhabditis elegans, the dsRBP RDE-4 acts with Dicer during the initiation of RNAi, when long dsRNA is cleaved to small interfering RNAs (siRNAs). RDE-4 is not sequence-specific, however, RDE-4 binds with higher affinity to long dsRNA. Interestingly, RDE- 4 is the only protein that exhibits micromolar affinity for dsRNA in the absence of co-operativity. In addition, RDE-4 is a homodimer in solution. <ref name=first>DOI: 10.1261/rna.2338706</ref> (Yellow Citation)<br />
<br />
<Structure load='2LTS' size='350' frame='true' align='right' caption='Structure of RDE-4 (pdb code 2LTS)'/><br />
<br />
<Structure load='2LTR' size='350' frame='true' align='right' caption='Structure of RDE-4 (pdb code 2LTR)'/><br />
<br />
== Structure of RDE-4 ==<br />
<br />
RDE-4 has five major regions: an <scene name='79/798389/N-terminal_of_rde-4/1'>N-terminal region (residues 1-43)</scene>, two dsRBDs (residues 44–108 and 170–235), a <scene name='79/798389/Long_linker_of_rde-4/1'>Long Linker (residues 109-169)</scene>, and a C-terminal domain (residues 236–385). Amino acid residues 1–32 and 136–151 adopt a random coiled structure and do not make any contact within itself or with the rest of the structure. Parts of the linker, residues 109–135 and 152–169, assume an α-helix and an extended loop structure, respectively. There is not any long-range nuclear Overhauser effect information between regions 1–135 and 152– 243, suggesting that the unstructured part of the linker 136–151 separates both of these regions. The region encompassed by 236–243 folds into an α-helix. The amino acid regions 44–108 and 170–235 form dsRBD1 and dsRBD2, respectively. The hydrophobic residues Leu45, Val47, Leu48, Val55, Trp62, Met73, Leu75, Leu77, Ile80, Val82, Leu101, and Val105 stabilize dsRBD1, and Val171, Leu174, Leu183, Val201, Met205, Met227, and Leu232 form the core of dsRBD2. Apart from the canonical dsRBD fold, there are additional secondary-structural elements that are observed in both RDE-4D1 and RDE-4D2. <ref name=second>DOI:DOI: 10.1042/BJ20131347</ref> (Purple citation)<br />
<br />
== Function ==<br />
<br />
In C. elegans, exogenous dsRNA is detected and bound by RDE-4, which stimulates dicer activity. RDE-4 initiates the siRNA pathway by binding to long dsRNA and assisting Dcr-1, a Dicer1 homologue, to facilitate siRNA production. RDE-4 interacts with Dcr-1, RDE-1, DRH-1 (Dicer-related helicase 1) and long dsRNA, which thereby suggests that RDE-4 is required only for the initiation of the RNAi pathway to generate siRNA, although high levels of dsRNA abrogate RDE-4’s role in the RNAi initiation. RDE-4 co- operatively binds to long dsRNA with nanomolar affinity, but exhibits micromolar affinity for short dsRNA, and the enhanced affinity arises from binding of several RDE-4 molecules to a single long dsRNA. <ref name=third>DOI: 10.1016/j.jmb.2008.10.002</ref> (Blue citation) The C-terminal region of RDE-4 is necessary and sufficient to induce homodimerization RDE-4 linker– dsRBD2 are absolutely essential in C. elegans gene silencing and RDE-4 dsRBD1 and the C-terminal domain do not have any primary role in vivo. RDE-4 recognizes the A-form of nucleic acid structure adopted by dsRNA and the interaction is sequence-independent.<ref name=second/>(Purple citation) RDE-4 dimerization is important for the assembly of active RDE-4/Dicer complexes via one of two proposed scenarios, proper Dicer recruitment to dsRNA, or facilitating Dicer dimerization. <ref name=first/> (Yellow citation)<br />
<br />
<br />
This is a sample scene created with SAT to <scene name="/12/3456/Sample/1">color</scene> by Group, and another to make <scene name="/12/3456/Sample/2">a transparent representation</scene> of the protein. You can make your own scenes on SAT starting from scratch or loading and editing one of these sample scenes.<br />
<br />
</StructureSection><br />
== References ==<br />
<references/></div>Wally Novakhttps://proteopedia.org/index.php?title=Rde_4_sandbox&diff=2955040Rde 4 sandbox2018-10-09T02:34:20Z<p>Wally Novak: </p>
<hr />
<div>==Introduction==<br />
<br />
RDE-4 was identified through a genome-wide screening as a gene responsible for greatly reducing or abolishing RNAi in C. elegans. In organisms ranging from Arabidopsis to humans, Dicer requires dsRNA-binding proteins (dsRBPs) to carry out its roles in RNA interference (RNAi) and micro-RNA (miRNA) processing. In Caenorhabditis elegans, the dsRBP RDE-4 acts with Dicer during the initiation of RNAi, when long dsRNA is cleaved to small interfering RNAs (siRNAs). RDE-4 is not sequence-specific, however, RDE-4 binds with higher affinity to long dsRNA. Interestingly, RDE- 4 is the only protein that exhibits micromolar affinity for dsRNA in the absence of co-operativity. In addition, RDE-4 is a homodimer in solution. <ref name=first>DOI: 10.1261/rna.2338706</ref> (Yellow Citation)<br />
<br />
<Structure load='2LTR' size='350' frame='true' align='right' caption='Structure of RDE-4 (pdb code 2LTR)'/> <br />
<br />
<Structure load='2LTS' size='350' frame='true' align='right' caption='Structure of RDE-4 (pdb code 2LTS)'/><br />
<br />
== Structure of RDE-4 ==<br />
<br />
RDE-4 has five major regions: an <scene name='79/798389/N-terminal_of_rde-4/1'>N-terminal region (residues 1-43)</scene>, two dsRBDs (residues 44–108 and 170–235), a <scene name='79/798389/Long_linker_of_rde-4/1'>Long Linker (residues 109-169)</scene>, and a C-terminal domain (residues 236–385). Amino acid residues 1–32 and 136–151 adopt a random coiled structure and do not make any contact within itself or with the rest of the structure. Parts of the linker, residues 109–135 and 152–169, assume an α-helix and an extended loop structure, respectively. There is not any long-range nuclear Overhauser effect information between regions 1–135 and 152– 243, suggesting that the unstructured part of the linker 136–151 separates both of these regions. The region encompassed by 236–243 folds into an α-helix. The amino acid regions 44–108 and 170–235 form dsRBD1 and dsRBD2, respectively. The hydrophobic residues Leu45, Val47, Leu48, Val55, Trp62, Met73, Leu75, Leu77, Ile80, Val82, Leu101, and Val105 stabilize dsRBD1, and Val171, Leu174, Leu183, Val201, Met205, Met227, and Leu232 form the core of dsRBD2. Apart from the canonical dsRBD fold, there are additional secondary-structural elements that are observed in both RDE-4D1 and RDE-4D2. <ref name=second>DOI:DOI: 10.1042/BJ20131347</ref> (Purple citation)<br />
<br />
== Function ==<br />
<br />
In C. elegans, exogenous dsRNA is detected and bound by RDE-4, which stimulates dicer activity. RDE-4 initiates the siRNA pathway by binding to long dsRNA and assisting Dcr-1, a Dicer1 homologue, to facilitate siRNA production. RDE-4 interacts with Dcr-1, RDE-1, DRH-1 (Dicer-related helicase 1) and long dsRNA, which thereby suggests that RDE-4 is required only for the initiation of the RNAi pathway to generate siRNA, although high levels of dsRNA abrogate RDE-4’s role in the RNAi initiation. RDE-4 co- operatively binds to long dsRNA with nanomolar affinity, but exhibits micromolar affinity for short dsRNA, and the enhanced affinity arises from binding of several RDE-4 molecules to a single long dsRNA. <ref name=third>DOI: 10.1016/j.jmb.2008.10.002</ref> (Blue citation) The C-terminal region of RDE-4 is necessary and sufficient to induce homodimerization RDE-4 linker– dsRBD2 are absolutely essential in C. elegans gene silencing and RDE-4 dsRBD1 and the C-terminal domain do not have any primary role in vivo. RDE-4 recognizes the A-form of nucleic acid structure adopted by dsRNA and the interaction is sequence-independent.<ref name=second/>(Purple citation) RDE-4 dimerization is important for the assembly of active RDE-4/Dicer complexes via one of two proposed scenarios, proper Dicer recruitment to dsRNA, or facilitating Dicer dimerization. <ref name=first/> (Yellow citation)<br />
<br />
<br />
This is a sample scene created with SAT to <scene name="/12/3456/Sample/1">color</scene> by Group, and another to make <scene name="/12/3456/Sample/2">a transparent representation</scene> of the protein. You can make your own scenes on SAT starting from scratch or loading and editing one of these sample scenes.<br />
<br />
</StructureSection><br />
== References ==<br />
<references/></div>Wally Novakhttps://proteopedia.org/index.php?title=Sandbox_RDE-1&diff=2955039Sandbox RDE-12018-10-09T02:23:02Z<p>Wally Novak: </p>
<hr />
<div>== Introduction ==<br />
<Structure load='4krf' size='350' frame='true' align='right' caption='Protein length: 1020 aa, Mass: 118,804 Da' scene='Insert optional scene name here' /><br />
The rde-1 gene is a member of the ''Argonaute'' gene family. Proteins from "Argonaute" family form an evolutionarily conserved family whose members silence gene expression in pathways such as RNA interference (RNAi). Argonaute family proteins can be divided into two types, AGO and Piwi proteins, depending on the small RNA they bonded to. Both types of Argonaute proteins bind 21–35 nucleotide-long small RNA guides whose sequence identifies the genes to be silenced.<ref name=four/><br />
'''RDE-1''' (RNAi-DEfective 1), a primary Argonaute protein, is required for RNA-mediated interference in ''Caenorhabditis elegans''; thus, it is also known as RNAi promoting factor. Its gene locus was first characterized in ''C. elegans'' mutants resistant to RNAi, and was found to be a member of the Piwi gene family that includes plant, Drosophila, and vertebrate homologs.<ref name=two>PMID: 10535731</ref><br />
<br />
== Structure ==<br />
All "Argonaute"/Piwi proteins possess three primary domains forming a crescent-shaped base: the PAZ, MID, and PIWI domains. The amino-terminal PAZ domain uses its oligonucleotide-binding (OB) fold to secure the 3′ end of the small RNA guide strand to ''Argonaute'' protein. A conserved hydrophobic cavity within the PAZ domain recognizes the characteristic two-nucleotide, 3′ overhanging end of the guide-passenger siRNA generated by Dicer. The MID domain anchors the 5′ monophosphate of a siRNA to the ''Argonaute'' protein, securing the guide through multiple cycles of target cleavage. In vitro, studies suggest that 5′ phosphate binding helps align the small RNA on the surface of "Argonaute" protein, ensuring that the correct bond of the target is positioned in the endonuclease active site.<ref name=four>PMID: 21683893</ref> In other words, the PAZ and MID domains orient and anchor the double-stranded siRNA by binding to the 3’ and 5’ termini, respectively, leaving the internal nucleotides accessible for base pairing.<ref name=four/><ref name=three>PMID:15284453</ref> The carboxy-terminal PIWI domain resembles nuclease RNase H in which it folds into an RNase H-like structure. This domain contains three conserved amino acids, aspartate-aspartate-histidine, that form a catalytic triad "DDH".<ref name=three/> The crystal structure of RDE-1 has not been fully elucidated, but can be assumed to closely resemble its human homologs. The full length of RDE-1 protein is 1020 amino acids (aa)<ref name=two/> in which about 110 of those aa makes up the PAZ domain and 300 aa makes up the PIWI domain.<ref> DOI: 10.1016/s0968-0004(00)01641-8</ref><br />
<br />
== Importance/Function ==<br />
[[Image:RDE-1 Proposed Fxn.png|300px||left|thumb|Proposed RNAi pathway for exogenous trigger dsRNA in C. elegans.<ref>https://en.wikipedia.org/wiki/RDE-1#/media/File:Exogenous_RNAi_Pathway_in_C._elegans,_edited.svg</ref>]] '''RDE-1''' is not required for the initial processing of the trigger RNA into siRNAs in RNAi, but it is required in the effector step. The trigger dsRNA is bound by RDE-4 onto a '''Dicer''' complex and this complex cleaves the dsRNA into 21-25nt primary siRNA. The siRNA binds to RDE-1 promoting the formation of the RNA-induced silencing complex (RISC) and RDE-1 shuttles the siRNA to that effector complex. The RNase H activity in PIWI domain in RDE-1 facilitates siRNA maturation, cleaving siRNA into a single stranded siRNA or guide RNA while RISC is activated when ATP is added to the complex and utilized the guide RNA on RDE-1 to base pair with the target transcript. The activation of RISC promotes the recruitment of RNA-dependent RNA polymerase (RdRP) which triggers the amplification of the secondary siRNAs to exhibit target transcript degradation.<br />
<br />
== Disease ==<br />
<br />
== Relevance ==<br />
<br />
<br />
<br />
This is a sample scene created with SAT to <scene name="/12/3456/Sample/1">color</scene> by Group, and another to make <scene name="/12/3456/Sample/2">a transparent representation</scene> of the protein. You can make your own scenes on SAT starting from scratch or loading and editing one of these sample scenes.<br />
<br />
</StructureSection><br />
<br />
== References ==<br />
<references/></div>Wally Novakhttps://proteopedia.org/index.php?title=Rde_4_sandbox&diff=2955038Rde 4 sandbox2018-10-09T02:21:52Z<p>Wally Novak: </p>
<hr />
<div>==Introduction==<br />
<br />
RDE-4 was identified through a genome-wide screening as a gene responsible for greatly reducing or abolishing RNAi in C. elegans. In organisms ranging from Arabidopsis to humans, Dicer requires dsRNA-binding proteins (dsRBPs) to carry out its roles in RNA interference (RNAi) and micro-RNA (miRNA) processing. In Caenorhabditis elegans, the dsRBP RDE-4 acts with Dicer during the initiation of RNAi, when long dsRNA is cleaved to small interfering RNAs (siRNAs). RDE-4 is not sequence-specific, however, RDE-4 binds with higher affinity to long dsRNA. Interestingly, RDE- 4 is the only protein that exhibits micromolar affinity for dsRNA in the absence of co-operativity. In addition, RDE-4 is a homodimer in solution. <ref name=first>DOI: 10.1261/rna.2338706</ref> (Yellow Citation)<br />
<br />
<Structure load='2LTR' size='350' frame='true' align='right' caption='Structure of RDE-4 (pdb code 2LTR) scene='Insert optional scene name here' /> <br />
<br />
== Structure of RDE-4 ==<br />
<br />
RDE-4 has five major regions: an <scene name='79/798389/N-terminal_of_rde-4/1'>N-terminal region (residues 1-43)</scene>, two dsRBDs (residues 44–108 and 170–235), a <scene name='79/798389/Long_linker_of_rde-4/1'>Long Linker (residues 109-169)</scene>, and a C-terminal domain (residues 236–385). Amino acid residues 1–32 and 136–151 adopt a random coiled structure and do not make any contact within itself or with the rest of the structure. Parts of the linker, residues 109–135 and 152–169, assume an α-helix and an extended loop structure, respectively. There is not any long-range nuclear Overhauser effect information between regions 1–135 and 152– 243, suggesting that the unstructured part of the linker 136–151 separates both of these regions. The region encompassed by 236–243 folds into an α-helix. The amino acid regions 44–108 and 170–235 form dsRBD1 and dsRBD2, respectively. The hydrophobic residues Leu45, Val47, Leu48, Val55, Trp62, Met73, Leu75, Leu77, Ile80, Val82, Leu101, and Val105 stabilize dsRBD1, and Val171, Leu174, Leu183, Val201, Met205, Met227, and Leu232 form the core of dsRBD2. Apart from the canonical dsRBD fold, there are additional secondary-structural elements that are observed in both RDE-4D1 and RDE-4D2. <ref name=second>DOI:DOI: 10.1042/BJ20131347</ref> (Purple citation)<br />
<br />
== Function ==<br />
<br />
In C. elegans, exogenous dsRNA is detected and bound by RDE-4, which stimulates dicer activity. RDE-4 initiates the siRNA pathway by binding to long dsRNA and assisting Dcr-1, a Dicer1 homologue, to facilitate siRNA production. RDE-4 interacts with Dcr-1, RDE-1, DRH-1 (Dicer-related helicase 1) and long dsRNA, which thereby suggests that RDE-4 is required only for the initiation of the RNAi pathway to generate siRNA, although high levels of dsRNA abrogate RDE-4’s role in the RNAi initiation. RDE-4 co- operatively binds to long dsRNA with nanomolar affinity, but exhibits micromolar affinity for short dsRNA, and the enhanced affinity arises from binding of several RDE-4 molecules to a single long dsRNA. <ref name=third>DOI: 10.1016/j.jmb.2008.10.002</ref> (Blue citation) The C-terminal region of RDE-4 is necessary and sufficient to induce homodimerization RDE-4 linker– dsRBD2 are absolutely essential in C. elegans gene silencing and RDE-4 dsRBD1 and the C-terminal domain do not have any primary role in vivo. RDE-4 recognizes the A-form of nucleic acid structure adopted by dsRNA and the interaction is sequence-independent.<ref name=second/>(Purple citation) RDE-4 dimerization is important for the assembly of active RDE-4/Dicer complexes via one of two proposed scenarios, proper Dicer recruitment to dsRNA, or facilitating Dicer dimerization. <ref name=first/> (Yellow citation)<br />
<br />
<br />
This is a sample scene created with SAT to <scene name="/12/3456/Sample/1">color</scene> by Group, and another to make <scene name="/12/3456/Sample/2">a transparent representation</scene> of the protein. You can make your own scenes on SAT starting from scratch or loading and editing one of these sample scenes.<br />
<br />
</StructureSection><br />
== References ==<br />
<references/></div>Wally Novakhttps://proteopedia.org/index.php?title=User:Wally_Novak/Sandbox_Whitaker&diff=2955037User:Wally Novak/Sandbox Whitaker2018-10-09T02:19:31Z<p>Wally Novak: </p>
<hr />
<div>==U5 snRNP==<br />
<StructureSection load='1stp' size='340' side='right' caption='Human Spliceosomal Protein U5-15kD'><br />
==Function==<br />
===General Overview===<br />
The U5 snRNP consists of several proteins which are highly conserved from yeast to humans. These proteins consist of Prp8, Snu114, Brr2, Prp28, Snu40/52K, and Dib1<ref>PMID:11720284</ref>, however, only Prp8, Snu114, and Brr2 remain in complex with the U5 snRNA while the spliceosome is activated <ref name="Irish">PMID:23354046</ref>. These proteins are necessary for the remodeling of the spliceosome and may play a role in maintaining catalytic activity at the active center of the spliceosome <ref name="Irish" />. Assembly of the U5 snRNP is followed by the formation of the U4/U6.U5 tri-snRNP which is the last remaining complex necessary to form the complete spliceosome and is therefore vital for its function. <br />
===U5 snRNA===<br />
The U5 snRNA interacts directly with the 5’ exon before the first step of splicing and with the 5’ and 3’ exons following the first step of splicing <ref name="Keefe">PMID:9430647</ref>. These U5–exon interactions are essential for tethering and aligning the exons for ligation during the second step of splicing <ref name="Keefe" />.<br />
===Brr2===<br />
<Structure load='3hib' size='400' frame='true' align='right' caption='Brr2="/><br />
Brr2 is an ATPase required for unwinding U4/U6 base-pairing before the first step of splicing and for unwinding U2/U6 base-pairing after the second step of splicing <ref>PMID:20659012</ref>. These conformational changes catalyzed by Brr2 are essential for the progression of the spliceosome cycle. <br />
===Prp8===<br />
♣Structure load='3e66' size='400' frame='true' align='right' caption='Prp8'/♣<br />
Prp8 is believed to be the master regulator of the splicing cycle by regulating the spliceosomal DExD/H-box RNA dependent ATPases, specifically Brr2 <ref>PMID:11017191</ref>. Consistent with this regulatory role it has been shown that the C-terminus of Prp8 activates Brr2 helicase function and inhibits Brr20 s U4/U6-dependent ATPase activity in vitro <ref>PMID:19098916</ref>. Structural studies of the Prp8 C-terminus have identified an RNase H-like domain within Prp8 and it has been proposed that this RNase H-domain may form the active site of the spliceosome <ref name="Irish" />.<br />
===Snu114===<br />
Snu114 is the only GTPase associated with the spliceosome and it has multiple contacts with Prp8 as well as U5 snRNA and Brr2 but its role in spliceosome function is not yet well-defined. <ref name="Irish" />.<br />
==Structural Highlights==<br />
Four sequences are highly conserved within the U5 snRNA and it is believed that these sequences play a primary role in the proteins binding capabilities. The four regions are the Stem-loop 1, Internal loop 1, Stem-closing internal loop 1, and Sm protein binding site <ref name="Frank">PMID:8114748</ref>. Stem-loop 1 spans nucleotides 84 to 110 (S. cerevisiae) and consists of a highly conserved 11 nucleotide block with complementary sequences on either side <ref name="Frank" />. These complementary sequences enable local base-pairing to form the stem of the loop <ref name="Frank" />. The U5 snRNA stem-loop 1 is necessary for tethering and aligning exons for ligation during the second step of splicing. The U5 snRNA stem 1 and internal loop 1 (IL1) are also highly conserved between yeast and humans, both in size and in structure <ref name="Frank" />. The U5 snRNA IL1 was found to be the most important region for the association of Prp8, Snu114 and Brr2 with the U5 snRNA <ref>PMID:23857713</ref>. Genetic analysis also identified the U5 snRNA IL1 as being important for Brr2 function <ref name="Frank" />. Overall, the U5 snRNA IL1 appears to be critical for the association of the U5 snRNP proteins Prp8, Snu114 and Brr2 with the U5 snRNA to form the U5 snRNP <ref name="Frank" />. <br />
===Brr2 Structural Highlights===<br />
♣Structure load='3hib' size='400' frame='true' align='right' caption='Brr2'/♣<br />
Recent structural analysis of Brr2 has revealed that part of the Sec63 like cassettes of the Brr2 helicase domains resemble the DNA helicase Hel308, hinting at an RNA unwinding action of Brr2 similar to that of the DNA unwinding by helicase Hel308 <ref name="Irish" />. The activity of Brr2 is regulated by the guanine nucleotide state of Snu114 <ref name="Irish" />.<br />
===Prp8 Structural Highlights===<br />
♣Structure load='3e66' size='400' frame='true' align='right' caption='Prp8'/♣<br />
Prp8 is a 280 kDa protein component of the U5 snRNP and is also part of the U4/U6.U5 tri-snRNP <ref name="Irish" />. Prp8 forms a complex with the assembly factor Aar2 and Snu114 without the U5 snRNA in the cytoplasm, however, Aar2 is replaced with Brr2 in the nucleus <ref name="Irish" />.<br />
==Protein Interactions==<br />
The U5 snRNP is one of the five subunits which come together to form the spliceosome and is, therefore, a key protein for eukaryotic mRNA processing. U5 snRNP is a complex which consists of U5 snRNA, Brr2, Snu114, and Prp8. Prp8 also interacts with other proteins of the spliceosome, the snRNAs and extensively with the pre-mRNA <ref name="Irish" />. It has been shown that Prp8 crosslinks directly to the pre-mRNA 5’ splice site, the branch site, and the 3’ splice site as well as U5 and U6 snRNAs localizing it to the heart of the spliceosome <ref name="Irish" />. In yeast, Prp8 makes extensive direct contacts with the U5 snRNA including the highly conserved U5 loop 1 and IL1 <ref name="Irish" />.<br />
Following successful assembly of the U5 snRNP, a tri-snRNP consisting of U4, U5 and U6 is formed which is then incorporated into the active spliceosome. ♣Structure load='5gan' size='400' frame='true' align='right' caption='U4/U6.U5 tri-snRNP'/♣<br />
This is a sample scene created with SAT to <scene name="/12/3456/Sample/1">color</scene> by Group, and another to make <scene name="/12/3456/Sample/2">a transparent representation</scene> of the protein. You can make your own scenes on SAT starting from scratch or loading and editing one of these sample scenes.<br />
<br />
</StructureSection><br />
== References ==<br />
<references/></div>Wally Novak