Sandbox Reserved 194: Difference between revisions

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== Introduction ==

RNase A is an endonuclease that cleaves and breaks down RNA using acid base catalysis. RNase A has been a model protein for studies on the stability, folding and chemistry of proteins. ‘<ref>PMID:11848924</ref>’ It is also essential in protein regulation within the body due to its function of RNA degradation.

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[[Image:1RTAnew.png|thumb|left|280px|Thymidylic acid tetramer complexed with ribonuclease A]]

To determine the structural characteristics of RNA substrate binding to RNase A, X-ray crystallography was used to image inhibitory DNA tetramers bound to RNase A. DNA lacks the 2’OH essential to RNA cleavage, making the complex more conducive to crystallography. <scene name='Sandbox_Reserved_194/1rta_structure/2'>The complex</scene> between RNase A and <scene name='Sandbox_Reserved_194/1rta_just_dt/2'>thymidylic acid tetramer (d(pT)4)</scene> provides information about specificity of the binding pocket subunits, B0, B1, B2 and B3. Many interactions observed in this complex occur between amino acid residues and the nucleic acid backbone. Examples of these interactions include hydrogen bonding between <scene name='Sandbox_Reserved_194/1rta_structure_arg/5'> phosphate of T1 and Arg39</scene> as well as hydrogen bonding between the O5’ oxygen of the ribose of <scene name='Sandbox_Reserved_194/1rta_lys41/1'>T3 and Lys41</scene>. ‘<ref>PMID:1429575</ref>’

To determine the structural characteristics of RNA substrate binding to RNase A, X-ray crystallography was used to image inhibitory DNA tetramers bound to RNase A. DNA lacks the 2’OH essential to RNA cleavage, making the complex more conducive to crystallography. <scene name='Sandbox_Reserved_194/1rta_structure/2'>The complex</scene> between RNase A and <scene name='Sandbox_Reserved_194/1rta_just_dt/2'>thymidylic acid tetramer (d(pT)4)</scene> ([[1rta]]) provides information about specificity of the binding pocket subunits, B0, B1, B2 and B3. Many interactions observed in this complex occur between amino acid residues and the nucleic acid backbone. Examples of these interactions include hydrogen bonding between <scene name='Sandbox_Reserved_194/1rta_structure_arg/5'> phosphate of T1 and Arg39</scene> as well as hydrogen bonding between the O5’ oxygen of the ribose of <scene name='Sandbox_Reserved_194/1rta_lys41/1'>T3 and Lys41</scene>. ‘<ref>PMID:1429575</ref>’

[[Image:1RCNnew.png|thumb|left|280px|ApTpApApG complexed with ribonuclease A]]

Further binding pocket characterization was performed using <scene name='Sandbox_Reserved_194/1rcn_structure/3'>RNase A complexed with the oligonucleotide d(ApTpApApG)</scene>. This <scene name='Sandbox_Reserved_194/1rcn_just_dtda/3'>tetramer</scene> was important in determining the specificity of the binding sites of RNase A. In this complex, the B1 site is thought to exclusively bind to pyrimidine bases due to steric interactions and <scene name='Sandbox_Reserved_194/1rcn_thr45/1'>hydrogen bonding to Thr45d</scene>. When Thr45 was mutated to glycine, purines readily bound to the B1 site. ‘<ref>PMID: 8193116</ref>’ This interaction appears to be the driving force behind its inability to bind purines. While binding of other nucleobases to the B2 and B3 sites is possible, the imaging of this complex elucidated the preferences for adenosine bases at these two positions. In addition to its catalytic activity His119 has also been shown to be important in substrate specificity. This is due to the <scene name='Sandbox_Reserved_194/1rcn_his/10'>pi stacking between His119 and A3 </scene>. When this site was mutated, the affinity for a poly(A) substrate was decreased by 104-fold. ‘<ref>PMID: 21391696</ref>It also establishes hydrogen bonding between <scene name='Sandbox_Reserved_194/1rcn_hydrogen_bonding/10'>Asn71-A3, Gln69-A3 and Gln69-A4</scene>. ‘<ref>PMID:8063789</ref>’

Further binding pocket characterization was performed using <scene name='Sandbox_Reserved_194/1rcn_structure/3'>RNase A complexed with the oligonucleotide d(ApTpApApG)</scene> ([[1rcn]]). This <scene name='Sandbox_Reserved_194/1rcn_just_dtda/3'>tetramer</scene> was important in determining the specificity of the binding sites of RNase A. In this complex, the B1 site is thought to exclusively bind to pyrimidine bases due to steric interactions and <scene name='Sandbox_Reserved_194/1rcn_thr45/1'>hydrogen bonding to Thr45d</scene>. When Thr45 was mutated to glycine, purines readily bound to the B1 site. ‘<ref>PMID: 8193116</ref>’ This interaction appears to be the driving force behind its inability to bind purines. While binding of other nucleobases to the B2 and B3 sites is possible, the imaging of this complex elucidated the preferences for adenosine bases at these two positions. In addition to its catalytic activity His119 has also been shown to be important in substrate specificity. This is due to the <scene name='Sandbox_Reserved_194/1rcn_his/10'>pi stacking between His119 and A3 </scene>. When this site was mutated, the affinity for a poly(A) substrate was decreased by 104-fold. ‘<ref>PMID: 21391696</ref>It also establishes hydrogen bonding between <scene name='Sandbox_Reserved_194/1rcn_hydrogen_bonding/10'>Asn71-A3, Gln69-A3 and Gln69-A4</scene>. ‘<ref>PMID:8063789</ref>’

== Conclusion ==

Sandbox Reserved 194: Difference between revisions

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@@ Line 6: / Line 6: @@
 == Introduction ==
-<Structure load='1RTA' size='450' frame='true' align='right' caption='Ribonuclease A complexed with thymidylic acid tetramer and ApTpApApG showing pi stacking and hydrogen bonding ' scene='Sandbox_Reserved_194/1rta_structure/2'/>
+<Structure load='1RTA' size='450' frame='true' align='right' caption='Ribonuclease A complexed with thymidylic acid tetramer and ApTpApApG showing pi stacking and hydrogen bonding' scene='Sandbox_Reserved_194/1rta_structure/2'/>
 RNase A is an endonuclease that cleaves and breaks down RNA using acid base catalysis. RNase A has been a model protein for studies on the stability, folding and chemistry of proteins. ‘<ref>PMID:11848924</ref>’ It is also essential in protein regulation within the body due to its function of RNA degradation.
@@ Line 14: / Line 14: @@
 [[Image:1RTAnew.png|thumb|left|280px|Thymidylic acid tetramer complexed with ribonuclease A]]
-To determine the structural characteristics of RNA substrate binding to RNase A, X-ray crystallography was used to image inhibitory DNA tetramers bound to RNase A. DNA lacks the 2’OH essential to RNA cleavage, making the complex more conducive to crystallography. <scene name='Sandbox_Reserved_194/1rta_structure/2'>The complex</scene> between RNase A and <scene name='Sandbox_Reserved_194/1rta_just_dt/2'>thymidylic acid tetramer (d(pT)4)</scene> provides information about specificity of the binding pocket subunits, B0, B1, B2 and B3. Many interactions observed in this complex occur between amino acid residues and the nucleic acid backbone. Examples of these interactions include hydrogen bonding between <scene name='Sandbox_Reserved_194/1rta_structure_arg/5'> phosphate of T1 and Arg39</scene> as well as hydrogen bonding between the O5’ oxygen of the ribose of <scene name='Sandbox_Reserved_194/1rta_lys41/1'>T3 and Lys41</scene>. ‘<ref>PMID:1429575</ref>’
+To determine the structural characteristics of RNA substrate binding to RNase A, X-ray crystallography was used to image inhibitory DNA tetramers bound to RNase A. DNA lacks the 2’OH essential to RNA cleavage, making the complex more conducive to crystallography. <scene name='Sandbox_Reserved_194/1rta_structure/2'>The complex</scene> between RNase A and <scene name='Sandbox_Reserved_194/1rta_just_dt/2'>thymidylic acid tetramer (d(pT)4)</scene> ([[1rta]]) provides information about specificity of the binding pocket subunits, B0, B1, B2 and B3. Many interactions observed in this complex occur between amino acid residues and the nucleic acid backbone. Examples of these interactions include hydrogen bonding between <scene name='Sandbox_Reserved_194/1rta_structure_arg/5'> phosphate of T1 and Arg39</scene> as well as hydrogen bonding between the O5’ oxygen of the ribose of <scene name='Sandbox_Reserved_194/1rta_lys41/1'>T3 and Lys41</scene>. ‘<ref>PMID:1429575</ref>’
 [[Image:1RCNnew.png|thumb|left|280px|ApTpApApG complexed with ribonuclease A]]
-Further binding pocket characterization was performed using <scene name='Sandbox_Reserved_194/1rcn_structure/3'>RNase A complexed with the oligonucleotide d(ApTpApApG)</scene>. This <scene name='Sandbox_Reserved_194/1rcn_just_dtda/3'>tetramer</scene> was important in determining the specificity of the binding sites of RNase A. In this complex, the B1 site is thought to exclusively bind to pyrimidine bases due to steric interactions and <scene name='Sandbox_Reserved_194/1rcn_thr45/1'>hydrogen bonding to Thr45d</scene>. When Thr45 was mutated to glycine, purines readily bound to the B1 site. ‘<ref>PMID: 8193116</ref>’  This interaction appears to be the driving force behind its inability to bind purines. While binding of other nucleobases to the B2 and B3 sites is possible, the imaging of this complex elucidated the preferences for adenosine bases at these two positions. In addition to its catalytic activity His119 has also been shown to be important in substrate specificity. This is due to the <scene name='Sandbox_Reserved_194/1rcn_his/10'>pi stacking between His119 and A3 </scene>. When this site was mutated, the affinity for a poly(A) substrate was decreased by 104-fold. ‘<ref>PMID: 21391696</ref>It also establishes hydrogen bonding between <scene name='Sandbox_Reserved_194/1rcn_hydrogen_bonding/10'>Asn71-A3, Gln69-A3 and Gln69-A4</scene>. ‘<ref>PMID:8063789</ref>’
+Further binding pocket characterization was performed using <scene name='Sandbox_Reserved_194/1rcn_structure/3'>RNase A complexed with the oligonucleotide d(ApTpApApG)</scene> ([[1rcn]]). This <scene name='Sandbox_Reserved_194/1rcn_just_dtda/3'>tetramer</scene> was important in determining the specificity of the binding sites of RNase A. In this complex, the B1 site is thought to exclusively bind to pyrimidine bases due to steric interactions and <scene name='Sandbox_Reserved_194/1rcn_thr45/1'>hydrogen bonding to Thr45d</scene>. When Thr45 was mutated to glycine, purines readily bound to the B1 site. ‘<ref>PMID: 8193116</ref>’  This interaction appears to be the driving force behind its inability to bind purines. While binding of other nucleobases to the B2 and B3 sites is possible, the imaging of this complex elucidated the preferences for adenosine bases at these two positions. In addition to its catalytic activity His119 has also been shown to be important in substrate specificity. This is due to the <scene name='Sandbox_Reserved_194/1rcn_his/10'>pi stacking between His119 and A3 </scene>. When this site was mutated, the affinity for a poly(A) substrate was decreased by 104-fold. ‘<ref>PMID: 21391696</ref>It also establishes hydrogen bonding between <scene name='Sandbox_Reserved_194/1rcn_hydrogen_bonding/10'>Asn71-A3, Gln69-A3 and Gln69-A4</scene>. ‘<ref>PMID:8063789</ref>’
 == Conclusion ==