https://proteopedia.org/api.php?action=feedcontributions&feedformat=atom&user=Raphael+BilgerProteopedia - User contributions [en]2026-09-15T00:16:13ZUser contributionsMediaWiki 1.43.8https://proteopedia.org/index.php?title=Sandbox_666&diff=1338393Sandbox 6662011-12-30T13:09:00Z<p>Raphael Bilger: </p>
<hr />
<div>Hello, this Sandbox is reserved for a student project. It describes the structure of the endonuclese restriction enzyme '''Eco RI'''.<br />
<br />
<br />
<br />
<br />
== Presentation ==<br />
<br />
<br />
<br />
<br />
''Eco''RI is a type II restriction endonuclease. It recognizes and cleaves DNA on a specific palindromic sequence: GAATTC. ''Eco''RI has been extracted from strain R ''Escherichia coli'', a common bacterium, which populates the intestine of mammalians. In bacteria, restriction enzymes protect the cell by cutting foreign DNA from bacteriophages (specific bacterial viruses) in both strands. Bacterial DNA is protected by a specific methylation of ''Eco''RI recognition sequences.<br />
<br />
<br />
<br />
== Reaction ==<br />
<br />
<br />
<br />
<br />
''Eco''RI (E.C. 3.1.21.4) is a hydrolase and its substrate is a double-strand DNA molecule and two water molecules. For its catalytic activity, ''Eco''RI needs a cofactor, which is the divalent ion Mg<sup>2+</sup>. ''Eco''RI hydrolyses the phosphodiester bond between the guanylic and adenylic residues resulting in 5’-phosphate sticky ends, which are complementary.<br />
<br />
<br />
<br />
== Structure ==<br />
<br />
{{STRUCTURE_1eri| PDB=1eri | SCENE= | size='500'}} <br />
''Eco''RI is composed of two homodimers, so it has two identical subunits (Representation of one <scene name='Sandbox_666/Monomer_structure/4'>subunit</scene>) of 31 kDa, but it is possible to have homotetramers at high concentrations. The constitutive monomers are 276 amino acids long. ''Eco''RI and all the other restriction enzymes show a common structural core, which is a α/β domain. There are several non-contiguous structural elements, whose are involved in DNA recognition<ref name="A">Recognition and cleavage of DNA by type-II restriction endonucleases, Pingoud A. & Jeltsch A. Eur.J.Biochem. 246,1-22 (1997)</ref>. The constitutive subunits of ''Eco''RI are organized into a single α/β domain (five strands <scene name='Sandbox_666/B_sheet/1'>β</scene> sheet, which is surrounded by <scene name='Sandbox_666/Helix/1'>two α helices</scene> ). Four of these five β strands are parallel whereas the fourth (β4) is in an anti-parallel orientation to the others<ref name="B">Refinement of Eco RI endonuclease crystal structure: a revised protein chain tracing, Kim, Y.C., Grable, J.C., Love, R., Greene, P.J., Rosenberg, J.M., Journal: (1990) Science 249: 1307-1309</ref>. Four helices (two of each subunit) recognize the major groove and bring residues, whose interact with DNA bases and backbones.<br />
<br />
[[Image:Image1 EcoRI.png |thumb| The topology of EcoRI restriction endonuclease<ref name="A" />.× catalytically important amino acid residues, • amino acid residues in interactions with DNA, pink regions: dimerization contacts|left|385px]]<br />
In the old model, the N-terminal section of each subunit forms the inner arm, which wraps around the DNA molecule (the arm brings the DNA molecule to the catalytic cleft). In the recent model, the extended chain motif ('''Met<sup>137</sup> to Ala<sup>142</sup>''') is a segment of the extended polypeptide chain, which runs through the major groove of the DNA, roughly parallel to the DNA backbone<ref name="B" /> and forms the specific contacts of the enzymes to the DNA. The outer arm is composed of two minor β strands linked together by a loop (the outer arm is 14 amino acids long, four of these amino acids belong to the loop).<br />
<br />
<br />
The specific recognition of ''Eco''RI of the GAATTC sequence is mediated by twelve hydrogen bonds (six bonds per subunit) originating from α helical recognition modules. Three amino acids are responsible for the recognition: '''Arg<sup>200</sup>, Glu<sup>144</sup> and Arg<sup>145</sup>'''. These amino acids are shown in red <scene name='Sandbox_666/Grey_protein/3'>here</scene>. Each residue form two hydrogen bonds with Guanine and the adjacent Adenosine residues respectively. There are also two arms, whose establish contacts with DNA backbones outside the recognition sequences.<br />
<br />
<br />
There is one ß-strand parallel to the DNA backbone, which contains amino acid residues essential for catalysis (e.g.: residues engaged in phosphate contacts<ref name="A" />). The reaction is due to a catalytic sequence motif, which is found in most type II restriction endonucleases: the '''PD…(D/E)XK motif'''. For ''Eco''RI,<scene name='Sandbox_666/Catalytic_core/3'> this catalytic sequence</scene> is '''PD<sup>91</sup> …E<sup>111</sup>AK''' and the lysine residue is essential to the catalysis, but the proline residue is not important. This motif is also responsible for Mg<sup>2+</sup> binding (Asp90 and Glu111)<ref>Structure and function of type II restriction endonucleases<br />
Alfred Pingoud, Albert Jeltsch<br />
Nucleic Acids Res. 2001 September 15; 29(18): 3705–3727.<br />
PMCID: PMC55916</ref>.<br />
<br />
<br />
This binding by the major groove is due to the position of scissile phosphodiester bonds.<ref name="A" /><br />
<br />
{|<br />
| [[Image:Catalytic 1ERI.png | thumb | The catalytic core of Eco RI with the target DNA. The Amino acids responsable for the reaction are shown in red and the target phosphodiester bond between the residues G and A is shown in blue |left|400px]]<br />
| <br />
|}<br />
<br />
<br />
<br />
==Catalytic mechanism<ref name="A" />==<br />
<br />
<br />
The function of ''Eco''RI is to cleave infectious DNA before it is methylated or before it begins to cause damage in the bacterial cell. That’s why ''Eco''RI is able to bind non-specifically anywhere to the DNA and continuously scans the DNA in a linear diffusion process with an approximate rate of 7*10<sup>6</sup> bp.s<sup>-1</sup>. In this way, the protein targets very quickly its specific sequences and any recognition sites is forgotten. This linear diffusion is due to electrostatic interactions; thence the DNA is trapped by ''Eco''RI but can move easily. Indeed during non-specific binding, there is no direct interaction with the bases of the DNA but there are only five interactions between amino acid residues from each subunit and the DNA phosphate groups. Moreover around 110 water molecules are located into this complex.<br />
It was observed that the enzyme makes pauses when the site is very similar to the recognition site (affinity of the enzyme to these sites). When one of these sites or a methylated site are cut, the reactional process is very slow and takes place only in one strand of the DNA, so the cellular DNA ligase can repair the nicked DNA. <br />
<br />
<br />
During the formation of the specific complex, the majority of the water molecules and ions are released and more and more direct interactions between the enzyme and the DNA are formed: some water molecules are immobilized between the enzyme and the DNA and formed hydrogen bounds, whose are important for the recognition and catalysis. Metal divalent ions, Mg<sup>2+</sup>, play also an essential role in the recognition process. The two homodimeres of ''Eco''RI cooperate in the binding and cleavage of their substrate and are symmetrically bound to the DNA. The specific complex formation leads to conformational changes of the protein and the DNA, thus catalytic centers of the both subunits are activated and the both DNA strands are simultaneously cleaved. <br />
Specific binding bend the DNA by about 12° and involve interactions between ''Eco''RI and DNA bases and phosphodiester backbones over approximately 10-12 bp. Moreover the residues involve to binding could participate at the catalysis. The number of interactions between ''Eco''RI and the recognition sequence is maximal. <br />
The restriction enzymes are highly specific and highly cooperative.<br />
<br />
<br />
<br />
== Application of EcoRI in molecular biology ==<br />
<br />
<br />
Type II restriction endonuclease like ''Eco''RI are often used in molecular biology for their capacity to cut precisely DNA on specific restriction site. That makes them useful tools for gene cloning. By using two different restriction enzymes, it is possible to do directional cloning, which is very important if you want to insert a gene in an expression vector.<br />
<br />
<br />
<br />
== Links ==<br />
[http://www.rcsb.org/pdb/explore/explore.do?structureId=1ERI] 1ERI in the Protein Database (PDB)<br />
<br />
<br />
<br />
== References ==<br />
<references/><br />
<br />
<br />
<br />
<br />
== Contributors ==<br />
<br />
Raphaël BILGER, Virginie GROSBOILLOT</div>Raphael Bilgerhttps://proteopedia.org/index.php?title=Sandbox_666&diff=1338391Sandbox 6662011-12-30T13:08:33Z<p>Raphael Bilger: </p>
<hr />
<div>Hello, this Sandbox is reserved for a student project. It describes the structure of the endonuclese restriction enzyme '''Eco RI'''.<br />
<br />
<br />
<br />
<br />
== Presentation ==<br />
<br />
<br />
<br />
<br />
''Eco''RI is a type II restriction endonuclease. It recognizes and cleaves DNA on a specific palindromic sequence: GAATTC. ''Eco''RI has been extracted from strain R ''Escherichia coli'', a common bacterium, which populates the intestine of mammalians. In bacteria, restriction enzymes protect the cell by cutting foreign DNA from bacteriophages (specific bacterial viruses) in both strands. Bacterial DNA is protected by a specific methylation of ''Eco''RI recognition sequences.<br />
<br />
<br />
<br />
== Reaction ==<br />
<br />
<br />
<br />
<br />
''Eco''RI (E.C. 3.1.21.4) is a hydrolase and its substrate is a double-strand DNA molecule and two water molecules. For its catalytic activity, ''Eco''RI needs a cofactor, which is the divalent ion Mg<sup>2+</sup>. ''Eco''RI hydrolyses the phosphodiester bond between the guanylic and adenylic residues resulting in 5’-phosphate sticky ends, which are complementary.<br />
<br />
<br />
<br />
== Structure ==<br />
<br />
{{STRUCTURE_1eri| PDB=1eri | SCENE= | size='500'}} <br />
''Eco''RI is composed of two homodimers, so it has two identical subunits (Representation of one <scene name='Sandbox_666/Monomer_structure/4'>subunit</scene>) of 31 kDa, but it is possible to have homotetramers at high concentrations. The constitutive monomers are 276 amino acids long. ''Eco''RI and all the other restriction enzymes show a common structural core, which is a α/β domain. There are several non-contiguous structural elements, whose are involved in DNA recognition<ref name="A">Recognition and cleavage of DNA by type-II restriction endonucleases, Pingoud A. & Jeltsch A. Eur.J.Biochem. 246,1-22 (1997)</ref>. The constitutive subunits of ''Eco''RI are organized into a single α/β domain (five strands <scene name='Sandbox_666/B_sheet/1'>β</scene> sheet, which is surrounded by <scene name='Sandbox_666/Helix/1'>two α helices</scene> ). Four of these five β strands are parallel whereas the fourth (β4) is in an anti-parallel orientation to the others<ref name="B">Refinement of Eco RI endonuclease crystal structure: a revised protein chain tracing, Kim, Y.C., Grable, J.C., Love, R., Greene, P.J., Rosenberg, J.M., Journal: (1990) Science 249: 1307-1309</ref>. Four helices (two of each subunit) recognize the major groove and bring residues, whose interact with DNA bases and backbones.<br />
<br />
[[Image:Image1 EcoRI.png |thumb| The topology of EcoRI restriction endonuclease<ref name="A" />.× catalytically important amino acid residues, • amino acid residues in interactions with DNA, pink regions: dimerization contacts|left|385px]]<br />
In the old model, the N-terminal section of each subunit forms the inner arm, which wraps around the DNA molecule (the arm brings the DNA molecule to the catalytic cleft). In the recent model, the extended chain motif ('''Met<sup>137</sup> to Ala<sup>142</sup>''') is a segment of the extended polypeptide chain, which runs through the major groove of the DNA, roughly parallel to the DNA backbone<ref name="B" /> and forms the specific contacts of the enzymes to the DNA. The outer arm is composed of two minor β strands linked together by a loop (the outer arm is 14 amino acids long, four of these amino acids belong to the loop).<br />
<br />
<br />
The specific recognition of ''Eco''RI of the GAATTC sequence is mediated by twelve hydrogen bonds (six bonds per subunit) originating from α helical recognition modules. Three amino acids are responsible for the recognition: '''Arg<sup>200</sup>, Glu<sup>144</sup> and Arg<sup>145</sup>'''. These amino acids are shown in red <scene name='Sandbox_666/Grey_protein/3'>here</scene>. Each residue form two hydrogen bonds with Guanine and the adjacent Adenosine residues respectively. There are also two arms, whose establish contacts with DNA backbones outside the recognition sequences.<br />
<br />
<br />
There is one ß-strand parallel to the DNA backbone, which contains amino acid residues essential for catalysis (e.g.: residues engaged in phosphate contacts<ref name="A" />). The reaction is due to a catalytic sequence motif, which is found in most type II restriction endonucleases: the '''PD…(D/E)XK motif'''. For ''Eco''RI,<scene name='Sandbox_666/Catalytic_core/3'> this catalytic sequence</scene> is '''PD<sup>91</sup> …E<sup>111</sup>AK''' and the lysine residue is essential to the catalysis, but the proline residue is not important. This motif is also responsible for Mg<sup>2+</sup> binding (Asp90 and Glu111)<ref>Structure and function of type II restriction endonucleases<br />
Alfred Pingoud, Albert Jeltsch<br />
Nucleic Acids Res. 2001 September 15; 29(18): 3705–3727.<br />
PMCID: PMC55916</ref>.<br />
<br />
<br />
This binding by the major groove is due to the position of scissile phosphodiester bonds.<ref name="A" /><br />
<br />
{|<br />
| [[Image:Catalytic 1ERI.png | thumb | The catalytic core of Eco RI with the target DNA. The Amino acids responsable for the reaction are shown in red and the target phosphodiester bond between the residues G and A is shown in blue |left|400px]]<br />
| <br />
|}<br />
<br />
<br />
<br />
==Catalytic mechanism<ref name="A" />==<br />
<br />
<br />
The function of ''Eco''RI is to cleave infectious DNA before it is methylated or before it begins to cause damage in the bacterial cell. That’s why ''Eco''RI is able to bind non-specifically anywhere to the DNA and continuously scans the DNA in a linear diffusion process with an approximate rate of 7*10<sup>6</sup> bp.s<sup>-1</sup>. In this way, the protein targets very quickly its specific sequences and any recognition sites is forgotten. This linear diffusion is due to electrostatic interactions; thence the DNA is trapped by ''Eco''RI but can move easily. Indeed during non-specific binding, there is no direct interaction with the bases of the DNA but there are only five interactions between amino acid residues from each subunit and the DNA phosphate groups. Moreover around 110 water molecules are located into this complex.<br />
It was observed that the enzyme makes pauses when the site is very similar to the recognition site (affinity of the enzyme to these sites). When one of these sites or a methylated site are cut, the reactional process is very slow and takes place only in one strand of the DNA, so the cellular DNA ligase can repair the nicked DNA. <br />
<br />
<br />
During the formation of the specific complex, the majority of the water molecules and ions are released and more and more direct interactions between the enzyme and the DNA are formed: some water molecules are immobilized between the enzyme and the DNA and formed hydrogen bounds, whose are important for the recognition and catalysis. Metal divalent ions, Mg<sup>2+</sup>, play also an essential role in the recognition process. The two homodimeres of ''Eco''RI cooperate in the binding and cleavage of their substrate and are symmetrically bound to the DNA. The specific complex formation leads to conformational changes of the protein and the DNA, thus catalytic centers of the both subunits are activated and the both DNA strands are simultaneously cleaved. <br />
Specific binding bend the DNA by about 12° and involve interactions between ''Eco''RI and DNA bases and phosphodiester backbones over approximately 10-12 bp. Moreover the residues involve to binding could participate at the catalysis. The number of interactions between ''Eco''RI and the recognition sequence is maximal. <br />
The restriction enzymes are highly specific and highly cooperative.<br />
<br />
<br />
<br />
== Application of EcoRI in molecular biology ==<br />
<br />
<br />
Type II restriction endonuclease like ''Eco''RI are often used in molecular biology for their capacity to cut precisely DNA on specific restriction site. That makes them useful tools for gene cloning. By using two different restriction enzymes, it is possible to do directional cloning, which is very important if you want to insert a gene in an expression vector.<br />
<br />
<br />
<br />
== Links ==<br />
[http://www.rcsb.org/pdb/explore/explore.do?structureId=1ERI] 1ERI in the Protein Database (PDB)<br />
<br />
<br />
<br />
== References ==<br />
<references/><br />
<br />
<br />
<br />
<br />
== Contributor ==<br />
<br />
Raphaël BILGER, Virginie GROSBOILLOT</div>Raphael Bilgerhttps://proteopedia.org/index.php?title=Sandbox_666&diff=1338390Sandbox 6662011-12-30T13:06:55Z<p>Raphael Bilger: </p>
<hr />
<div>Hello this Sandbox is reserved for a student project. It describes the structure of the endonuclese restriction enzyme '''Eco RI'''.<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
== Presentation ==<br />
<br />
<br />
<br />
''Eco''RI is a type II restriction endonuclease. It recognizes and cleaves DNA on a specific palindromic sequence: GAATTC. ''Eco''RI has been extracted from strain R ''Escherichia coli'', a common bacterium, which populates the intestine of mammalians. In bacteria, restriction enzymes protect the cell by cutting foreign DNA from bacteriophages (specific bacterial viruses) in both strands. Bacterial DNA is protected by a specific methylation of ''Eco''RI recognition sequences.<br />
<br />
<br />
<br />
== Reaction ==<br />
<br />
<br />
<br />
''Eco''RI (E.C. 3.1.21.4) is a hydrolase and its substrate is a double-strand DNA molecule and two water molecules. For its catalytic activity, ''Eco''RI needs a cofactor, which is the divalent ion Mg<sup>2+</sup>. ''Eco''RI hydrolyses the phosphodiester bond between the guanylic and adenylic residues resulting in 5’-phosphate sticky ends, which are complementary.<br />
<br />
<br />
<br />
== Structure ==<br />
<br />
{{STRUCTURE_1eri| PDB=1eri | SCENE= | size='500'}} <br />
''Eco''RI is composed of two homodimers, so it has two identical subunits (Representation of one <scene name='Sandbox_666/Monomer_structure/4'>subunit</scene>) of 31 kDa, but it is possible to have homotetramers at high concentrations. The constitutive monomers are 276 amino acids long. ''Eco''RI and all the other restriction enzymes show a common structural core, which is a α/β domain. There are several non-contiguous structural elements, whose are involved in DNA recognition<ref name="A">Recognition and cleavage of DNA by type-II restriction endonucleases, Pingoud A. & Jeltsch A. Eur.J.Biochem. 246,1-22 (1997)</ref>. The constitutive subunits of ''Eco''RI are organized into a single α/β domain (five strands <scene name='Sandbox_666/B_sheet/1'>β</scene> sheet, which is surrounded by <scene name='Sandbox_666/Helix/1'>two α helices</scene> ). Four of these five β strands are parallel whereas the fourth (β4) is in an anti-parallel orientation to the others<ref name="B">Refinement of Eco RI endonuclease crystal structure: a revised protein chain tracing, Kim, Y.C., Grable, J.C., Love, R., Greene, P.J., Rosenberg, J.M., Journal: (1990) Science 249: 1307-1309</ref>. Four helices (two of each subunit) recognize the major groove and bring residues, whose interact with DNA bases and backbones.<br />
<br />
[[Image:Image1 EcoRI.png |thumb| The topology of EcoRI restriction endonuclease<ref name="A" />.× catalytically important amino acid residues, • amino acid residues in interactions with DNA, pink regions: dimerization contacts|left|385px]]<br />
In the old model, the N-terminal section of each subunit forms the inner arm, which wraps around the DNA molecule (the arm brings the DNA molecule to the catalytic cleft). In the recent model, the extended chain motif ('''Met<sup>137</sup> to Ala<sup>142</sup>''') is a segment of the extended polypeptide chain, which runs through the major groove of the DNA, roughly parallel to the DNA backbone<ref name="B" /> and forms the specific contacts of the enzymes to the DNA. The outer arm is composed of two minor β strands linked together by a loop (the outer arm is 14 amino acids long, four of these amino acids belong to the loop).<br />
<br />
<br />
The specific recognition of ''Eco''RI of the GAATTC sequence is mediated by twelve hydrogen bonds (six bonds per subunit) originating from α helical recognition modules. Three amino acids are responsible for the recognition: '''Arg<sup>200</sup>, Glu<sup>144</sup> and Arg<sup>145</sup>'''. These amino acids are shown in red <scene name='Sandbox_666/Grey_protein/3'>here</scene>. Each residue form two hydrogen bonds with Guanine and the adjacent Adenosine residues respectively. There are also two arms, whose establish contacts with DNA backbones outside the recognition sequences.<br />
<br />
<br />
There is one ß-strand parallel to the DNA backbone, which contains amino acid residues essential for catalysis (e.g.: residues engaged in phosphate contacts<ref name="A" />). The reaction is due to a catalytic sequence motif, which is found in most type II restriction endonucleases: the '''PD…(D/E)XK motif'''. For ''Eco''RI,<scene name='Sandbox_666/Catalytic_core/3'> this catalytic sequence</scene> is '''PD<sup>91</sup> …E<sup>111</sup>AK''' and the lysine residue is essential to the catalysis, but the proline residue is not important. This motif is also responsible for Mg<sup>2+</sup> binding (Asp90 and Glu111)<ref>Structure and function of type II restriction endonucleases<br />
Alfred Pingoud, Albert Jeltsch<br />
Nucleic Acids Res. 2001 September 15; 29(18): 3705–3727.<br />
PMCID: PMC55916</ref>.<br />
<br />
<br />
This binding by the major groove is due to the position of scissile phosphodiester bonds.<ref name="A" /><br />
<br />
{|<br />
| [[Image:Catalytic 1ERI.png | thumb | The catalytic core of Eco RI with the target DNA. The Amino acids responsable for the reaction are shown in red and the target phosphodiester bond between the residues G and A is shown in blue |left|400px]]<br />
| <br />
|}<br />
<br />
<br />
<br />
==Catalytic mechanism<ref name="A" />==<br />
<br />
<br />
The function of ''Eco''RI is to cleave infectious DNA before it is methylated or before it begins to cause damage in the bacterial cell. That’s why ''Eco''RI is able to bind non-specifically anywhere to the DNA and continuously scans the DNA in a linear diffusion process with an approximate rate of 7*10<sup>6</sup> bp.s<sup>-1</sup>. In this way, the protein targets very quickly its specific sequences and any recognition sites is forgotten. This linear diffusion is due to electrostatic interactions; thence the DNA is trapped by ''Eco''RI but can move easily. Indeed during non-specific binding, there is no direct interaction with the bases of the DNA but there are only five interactions between amino acid residues from each subunit and the DNA phosphate groups. Moreover around 110 water molecules are located into this complex.<br />
It was observed that the enzyme makes pauses when the site is very similar to the recognition site (affinity of the enzyme to these sites). When one of these sites or a methylated site are cut, the reactional process is very slow and takes place only in one strand of the DNA, so the cellular DNA ligase can repair the nicked DNA. <br />
<br />
<br />
During the formation of the specific complex, the majority of the water molecules and ions are released and more and more direct interactions between the enzyme and the DNA are formed: some water molecules are immobilized between the enzyme and the DNA and formed hydrogen bounds, whose are important for the recognition and catalysis. Metal divalent ions, Mg<sup>2+</sup>, play also an essential role in the recognition process. The two homodimeres of ''Eco''RI cooperate in the binding and cleavage of their substrate and are symmetrically bound to the DNA. The specific complex formation leads to conformational changes of the protein and the DNA, thus catalytic centers of the both subunits are activated and the both DNA strands are simultaneously cleaved. <br />
Specific binding bend the DNA by about 12° and involve interactions between ''Eco''RI and DNA bases and phosphodiester backbones over approximately 10-12 bp. Moreover the residues involve to binding could participate at the catalysis. The number of interactions between ''Eco''RI and the recognition sequence is maximal. <br />
The restriction enzymes are highly specific and highly cooperative.<br />
<br />
<br />
<br />
== Application of EcoRI in molecular biology ==<br />
<br />
<br />
Type II restriction endonuclease like ''Eco''RI are often used in molecular biology for their capacity to cut precisely DNA on specific restriction site. That makes them useful tools for gene cloning. By using two different restriction enzymes, it is possible to do directional cloning, which is very important if you want to insert a gene in an expression vector.<br />
<br />
<br />
<br />
== Links ==<br />
[http://www.rcsb.org/pdb/explore/explore.do?structureId=1ERI] 1ERI in the Protein Database (PDB)<br />
<br />
<br />
<br />
== References ==<br />
<references/><br />
<br />
<br />
<br />
<br />
== Contributor ==<br />
<br />
Raphaël BILGER, Virginie GROSBOILLOT</div>Raphael Bilgerhttps://proteopedia.org/index.php?title=Sandbox_666&diff=1338274Sandbox 6662011-12-29T16:03:00Z<p>Raphael Bilger: </p>
<hr />
<div>Hello this Sandbox is reserved for a student project. It describes the structure of the endonuclese restriction enzyme '''Eco RI'''.<br />
<br />
== Presentation ==<br />
<br />
<br />
''Eco''RI is a type II restriction endonuclease. It recognizes and cleaves DNA on a specific palindromic sequence: GAATTC. ''Eco''RI has been extracted from strain R ''Escherichia coli'', a common bacterium, which populates the intestine of mammalians. In bacteria, restriction enzymes protect the cell by cutting foreign DNA from bacteriophages (specific bacterial viruses) in both strands. Bacterial DNA is protected by a specific methylation of ''Eco''RI recognition sequences.<br />
<br />
== Reaction ==<br />
<br />
<br />
''Eco''RI (E.C. 3.1.21.4) is a hydrolase and its substrate is a double-strand DNA molecule and two water molecules. For its catalytic activity, ''Eco''RI needs a cofactor, which is the divalent ion Mg<sup>2+</sup>. ''Eco''RI hydrolyses the phosphodiester bond between the guanylic and adenylic residues resulting in 5’-phosphate sticky ends, which are complementary.<br />
<br />
== Structure ==<br />
<br />
{{STRUCTURE_1eri| PDB=1eri | SCENE= | size='500'}} <br />
''Eco''RI is composed of two homodimers, so it has two identical subunits (Representation of one <scene name='Sandbox_666/Monomer_structure/4'>subunit</scene>) of 31 kDa, but it is possible to have homotetramers at high concentrations. The constitutive monomers are 276 amino acids long. ''Eco''RI and all the other restriction enzymes show a common structural core, which is a α/β domain. The constitutive subunits of ''Eco''RI are organized into a single α/β domain (five strands <scene name='Sandbox_666/B_sheet/1'>β</scene> sheet, which is surrounded by <scene name='Sandbox_666/Helix/1'>two α helices</scene> ). Four of these five β strands are parallel whereas the fourth (β4) is in an anti-parallel orientation to the others.<ref name="B">Refinement of Eco RI endonuclease crystal structure: a revised protein chain tracing.<br />
<br />
Kim, Y.C., Grable, J.C., Love, R., Greene, P.J., Rosenberg, J.M., <br />
<br />
Journal: (1990) Science 249: 1307-1309 </ref><br />
<br />
<br />
In the old model the N-terminal section of each subunit forms the inner arm which wraps around the DNA molecule (The arm brings the DNA molecule to the catalytic cleft.).The new chain tracing, based on new elements of electron density and a new interpretation that alters the assignment of specific amino acid residues to some of the original features<ref name="B" />. In the recent model,the inner arm is the extended chain motif ('''Met<sup>137</sup>to Ala<sup>142</sup>''') is a segment of extended polypeptide chain that runs through the major groove of the DNA, roughly parallel to the DNA backbone<ref name="B" />. The outer arm is composed of two minor β strands linked together by a loop (the outer arm is 14 amino acids long, four of these amino acids belong to the loop).<br />
<br />
<br />
The specific recognition of ''Eco''RI of the GAATTC sequence is mediated by twelve hydrogen bonds (six bonds per subunit) originating from α helical recognition modules. Three amino acids are responsible for the recognition:'''Arg<sup>200</sup>, Glu<sup>144</sup> and Arg<sup>145</sup>'''. These aminoacids are shown in red <scene name='Sandbox_666/Grey_protein/3'>here</scene> .Each residue form two hydrogen bonds with Guanine and the adjacent Adenosine residues respectively. <br />
<br />
<br />
The reaction is due to a catalytic sequence motif which is found in most type II restriction endonucleases: the '''PD…(D/E)XK motif'''. For ''Eco''RI,<scene name='Sandbox_666/Catalytic_core/3'>this catalytic sequence</scene> is '''PD<sup>91</sup> …E<sup>111</sup>AK''' and the lysine residue is essential to the catalysis, but the proline residue is not important. This motif is also responsible for Mg2+ binding(Asp90 and Glu111).<ref>Structure and function of type II restriction endonucleases<br />
Alfred Pingoud, Albert Jeltsch<br />
Nucleic Acids Res. 2001 September 15; 29(18): 3705–3727.<br />
PMCID: PMC55916</ref><br />
<br />
<br />
There are several non-contiguous structural elements, which are involved in DNA recognition<ref name="A" />:<br />
* Four helix (two of each subunit) recognize the major groove and bring residues, whose interact with DNA bases and backbones<br />
* An extended peptide chain running through the major groove forms the specific contacts of the enzymes to the DNA.<br />
* One ß-strand parallel to the DNA backbone, which contains amino acid residues essential for catalysis (e.g.: residues engaged in phosphate contacts)<br />
* Two arms, which establish contacts with DNA backbones outside the recognition sequences.<br />
<br />
This binding by the major groove is due to the position of scissile phosphodiester bonds.<ref name="A">Recognition and cleavage of DNA by type-II restriction endonucleases, Pingoud A. & Jeltsch A. Eur.J.Biochem. 246,1-22 (1997)</ref><br />
<br />
{|<br />
| [[Image:Catalytic 1ERI.png | thumb | The catalytic core of Eco RI with the target DNA|left|395px]]<br />
| [[Image:Image1 EcoRI.png |thumb| The topology of EcoRI restriction endonuclease<ref name="A" />.× catalytically important amino acid residues, • amino acid residues in interactions with DNA, pink regions: dimerization contacts|right|385px]]<br />
|}<br />
<br />
==Catalytic mechanism==<br />
<br />
<br />
The function of EcoRI is to cleave infectious DNA before it is methylated or before it begins to cause damage in the bacterial cell. That’s why EcoRI is able to bind non-specifically anywhere to the DNA and continuously scans the DNA in a linear diffusion process with an approximate rate of 7*106 bp.s-1. In this way, the protein targets very quickly its specific sequences and any recognition sites is forgotten. This linear diffusion is due to electrostatic interactions; thence the DNA is trapped by EcoRI but can move easily. Indeed during non-specific binding, there is no direct interaction with the bases of the DNA but there are only five interactions between amino acid residues from each subunit and the DNA phosphate groups. Moreover around 110 water molecules are located into this complex.<br />
<br />
It was observed that the enzyme makes pauses when the site is very similar to the recognition site (affinity of the enzyme to these sites). When one of these sites or a methylated site are cut, the reactional process is very slow and takes place only in one strand of the DNA, so the cellular DNA ligase can repair the nicked DNA. <br />
<br />
During the formation of the specific complex, the majority of the water molecules and ions are released and more and more direct interactions between the enzyme and the DNA are formed: some water molecules are immobilized between the enzyme and the DNA and formed hydrogen bounds, whose are important for the recognition and catalysis. Metal divalent ions, Mg2+, play also an essential role in the recognition process. The two homodimeres of EcoRI cooperate in the binding and cleavage of their substrate and are symmetrically bound to the DNA. The specific complex formation leads to conformational changes of the protein and the DNA, thus catalytic centers of the both subunits are activated and the both DNA strands are simultaneously cleaved. <br />
Specific binding bend the DNA by about 12° and involve interactions between EcoRI and DNA bases and phosphodiester backbones over approximately 10-12 bp. Moreover the residues involve to binding could participate at the catalysis. The number of interactions between EcoRI and the recognition sequence is maximal. <br />
The restriction enzymes are highly specific and highly cooperative.<ref name="A" /><br />
<br />
== Application of EcoRI in molecular biology ==<br />
<br />
<br />
Type II restriction endonuclease like ''Eco''RI are often used in molecular biology for their capacity to cut precisely DNA on specific restriction site. That makes them useful tools for gene cloning. By using two different restriction enzymes, it is possible to do directional cloning, which is very important if you want to insert a gene in an expression vector.<br />
<br />
== Links ==<br />
[http://www.rcsb.org/pdb/explore/explore.do?structureId=1ERI] 1ERI in the Protein Database (PDB)<br />
<br />
== References ==<br />
<references/> <br />
<br />
== Contributor ==<br />
<br />
Raphaël BILGER, Virginie GROSBOILLOT</div>Raphael Bilgerhttps://proteopedia.org/index.php?title=Sandbox_666&diff=1338242Sandbox 6662011-12-29T15:04:27Z<p>Raphael Bilger: </p>
<hr />
<div>Hello this Sandbox is reserved for a student project. It describes the structure of the endonuclese restriction enzyme '''Eco RI'''.<br />
<br />
== Presentation ==<br />
<br />
<br />
''Eco''RI is a type II restriction endonuclease. It recognizes and cleaves DNA on a specific palindromic sequence: GAATTC. ''Eco''RI has been extracted from strain R ''Escherichia coli'', a common bacterium, which populates the intestine of mammalians. In bacteria, restriction enzymes protect the cell by cutting foreign DNA from bacteriophages (specific bacterial viruses) in both strands. Bacterial DNA is protected by a specific methylation of ''Eco''RI recognition sequences.<br />
<br />
== Reaction ==<br />
<br />
<br />
''Eco''RI (E.C. 3.1.21.4) is a hydrolase and its substrate is a double-strand DNA molecule and two water molecules. For its catalytic activity, ''Eco''RI needs a cofactor, which is the divalent ion Mg<sup>2+</sup>. ''Eco''RI hydrolyses the phosphodiester bond between the guanylic and adenylic residues resulting in 5’-phosphate sticky ends, which are complementary.<br />
<br />
== Structure ==<br />
<br />
{{STRUCTURE_1eri| PDB=1eri | SCENE= | size='500'}} <br />
''Eco''RI is composed of two homodimers, so it has two identical subunits (Representation of one <scene name='Sandbox_666/Monomer_structure/4'>subunit</scene>) of 31 kDa, but it is possible to have homotetramers at high concentrations. The constitutive monomers are 276 amino acids long. ''Eco''RI and all the other restriction enzymes show a common structural core, which is a α/β domain. The constitutive subunits of ''Eco''RI are organized into a single α/β domain (five strands <scene name='Sandbox_666/B_sheet/1'>β</scene> sheet, which is surrounded by <scene name='Sandbox_666/Helix/1'>two α helices</scene> ). Four of these five β strands are parallel whereas the fourth (β4) is in an anti-parallel orientation to the others.<ref name="B">Refinement of Eco RI endonuclease crystal structure: a revised protein chain tracing.<br />
<br />
Kim, Y.C., Grable, J.C., Love, R., Greene, P.J., Rosenberg, J.M., <br />
<br />
Journal: (1990) Science 249: 1307-1309 </ref><br />
<br />
[[Image:Image1 EcoRI.png |thumb| The topology of EcoRI restriction endonuclease |left| 400px]] <br />
In the old model the N-terminal section of each subunit forms the inner arm which wraps around the DNA molecule (The arm brings the DNA molecule to the catalytic cleft.).The new chain tracing, based on new elements of electron density and a new interpretation that alters the assignment of specific amino acid residues to some of the original features<ref name="B" />. In the new model,the Inner arm is the extended chain motif (Met<sup>137</sup>to Ala<sup>142</sup>)is a segment of extended polypeptide chain that runs through the major groove of the DNA, roughly parallel to the DNA backbone<ref name="B" />. The outer arm is composed of two minor β strands linked together by a loop (the outer arm is 14 amino acids long, four of these amino acids belong to the loop).<br />
<br />
<br />
The specific recognition of ''Eco''RI of the GAATTC sequence is mediated by twelve hydrogen bonds (six bonds per subunit) originating from α helical recognition modules. Three amino acids are responsible for the recognition:Arg<sup>200</sup>, Glu<sup>144</sup> and Arg<sup>145</sup>. These aminoacids are shown in red <scene name='Sandbox_666/Grey_protein/3'>here</scene> .Each residue form two hydrogen bonds with Guanine and the adjacent Adenosine residues respectively. <br />
<br />
<br />
The reaction is due to a catalytic sequence motif which is found in most type II restriction endonucleases: the PD…(D/E)XK motif. For ''Eco''RI,<scene name='Sandbox_666/Catalytic_core/3'>this catalytic sequence</scene> is PD<sup>91</sup> …E<sup>111</sup>AK and the lysine residue is essential to the catalysis, but the proline residue is not important. This motif is also responsible for Mg2+ binding(Asp90 and Glu111).<ref>Structure and function of type II restriction endonucleases<br />
Alfred Pingoud, Albert Jeltsch<br />
Nucleic Acids Res. 2001 September 15; 29(18): 3705–3727.<br />
PMCID: PMC55916</ref><br />
<br />
<br />
There are several non-contiguous structural elements, which are involved in DNA recognition:<br />
* Four helix (two of each subunit) recognize the major groove and bring residues, whose interact with DNA bases and backbones<br />
* An extended peptide chain running through the major groove forms the specific contacts of the enzymes to the DNA.<br />
* One ß-strand parallel to the DNA backbone, which contains amino acid residues essential for catalysis (e.g.: residues engaged in phosphate contacts)<br />
* Two arms, which establish contacts with DNA backbones outside the recognition sequences.<br />
<br />
This binding by the major groove is due to the position of scissile phosphodiester bonds.<ref name="A">Recognition and cleavage of DNA by type-II restriction endonucleases, Pingoud A. & Jeltsch A. Eur.J.Biochem. 246,1-22 (1997)</ref><br />
<br />
[[Image:Catalytic 1ERI.png | thumb | The catalytic core of Eco RI with the target DNA|left|400px]]<br />
<br />
==Catalytic mechanism==<br />
<br />
<br />
The function of EcoRI is to cleave infectious DNA before it is methylated or before it begins to cause damage in the bacterial cell. That’s why EcoRI is able to bind non-specifically anywhere to the DNA and continuously scans the DNA in a linear diffusion process with an approximate rate of 7*106 bp.s-1. In this way, the protein targets very quickly its specific sequences and any recognition sites is forgotten. This linear diffusion is due to electrostatic interactions; thence the DNA is trapped by EcoRI but can move easily. Indeed during non-specific binding, there is no direct interaction with the bases of the DNA but there are only five interactions between amino acid residues from each subunit and the DNA phosphate groups. Moreover around 110 water molecules are located into this complex.<br />
<br />
It was observed that the enzyme makes pauses when the site is very similar to the recognition site (affinity of the enzyme to these sites). When one of these sites or a methylated site are cut, the reactional process is very slow and takes place only in one strand of the DNA, so the cellular DNA ligase can repair the nicked DNA. <br />
<br />
During the formation of the specific complex, the majority of the water molecules and ions are released and more and more direct interactions between the enzyme and the DNA are formed: some water molecules are immobilized between the enzyme and the DNA and formed hydrogen bounds, whose are important for the recognition and catalysis. Metal divalent ions, Mg2+, play also an essential role in the recognition process. The two homodimeres of EcoRI cooperate in the binding and cleavage of their substrate and are symmetrically bound to the DNA. The specific complex formation leads to conformational changes of the protein and the DNA, thus catalytic centers of the both subunits are activated and the both DNA strands are simultaneously cleaved. <br />
Specific binding bend the DNA by about 12° and involve interactions between EcoRI and DNA bases and phosphodiester backbones over approximately 10-12 bp. Moreover the residues involve to binding could participate at the catalysis. The number of interactions between EcoRI and the recognition sequence is maximal. <br />
The restriction enzymes are highly specific and highly cooperative.<ref name="A" /><br />
<br />
== Application of EcoRI in molecular biology ==<br />
<br />
<br />
Type II restriction endonuclease like ''Eco''RI are often used in molecular biology for their capacity to cut precisely DNA on specific restriction site. That makes them useful tools for gene cloning. By using two different restriction enzymes, it is possible to do directional cloning, which is very important if you want to insert a gene in an expression vector.<br />
<br />
== Links ==<br />
[http://www.rcsb.org/pdb/explore/explore.do?structureId=1ERI] 1ERI in the Protein Database (PDB)<br />
<br />
== References ==<br />
<references/> <br />
<br />
== Contributor ==<br />
<br />
Raphaël BILGER, Virginie GROSBOILLOT</div>Raphael Bilgerhttps://proteopedia.org/index.php?title=Sandbox_666&diff=1338234Sandbox 6662011-12-29T14:52:49Z<p>Raphael Bilger: </p>
<hr />
<div>Hello this Sandbox is reserved for a student project. It describes the structure of the endonuclese restriction enzyme '''Eco RI'''.<br />
<br />
== Presentation ==<br />
<br />
<br />
''Eco''RI is a type II restriction endonuclease. It recognizes and cleaves DNA on a specific palindromic sequence: GAATTC. ''Eco''RI has been extracted from strain R ''Escherichia coli'', a common bacterium, which populates the intestine of mammalians. In bacteria, restriction enzymes protect the cell by cutting foreign DNA from bacteriophages (specific bacterial viruses) in both strands. Bacterial DNA is protected by a specific methylation of ''Eco''RI recognition sequences.<br />
<br />
== Reaction ==<br />
<br />
<br />
''Eco''RI (E.C. 3.1.21.4) is a hydrolase and its substrate is a double-strand DNA molecule and two water molecules. For its catalytic activity, ''Eco''RI needs a cofactor, which is the divalent ion Mg<sup>2+</sup>. ''Eco''RI hydrolyses the phosphodiester bond between the guanylic and adenylic residues resulting in 5’-phosphate sticky ends, which are complementary.<br />
<br />
== Structure ==<br />
<br />
{{STRUCTURE_1eri| PDB=1eri | SCENE= | size='500'}} <br />
''Eco''RI is composed of two homodimers, so it has two identical subunits (Representation of one <scene name='Sandbox_666/Monomer_structure/4'>subunit</scene>) of 31 kDa, but it is possible to have homotetramers at high concentrations. The constitutive monomers are 276 amino acids long. ''Eco''RI and all the other restriction enzymes show a common structural core, which is a α/β domain. The constitutive subunits of ''Eco''RI are organized into a single α/β domain (five strands <scene name='Sandbox_666/B_sheet/1'>β</scene> sheet, which is surrounded by <scene name='Sandbox_666/Helix/1'>two α helices</scene> ). Four of these five β strands are parallel whereas the fourth (β4) is in an anti-parallel orientation to the others.<ref name="B">Refinement of ''Eco''RI endonuclease crystal structure: a revised protein chain tracing.<br />
Kim YC, Grable JC, Love R, Greene PJ, Rosenberg JM.</ref><br />
<br />
[[Image:Image1 EcoRI.png |thumb| The topology of EcoRI restriction endonuclease |left| 400px]] <br />
In the old model the N-terminal section of each subunit forms the inner arm which wraps around the DNA molecule (The arm brings the DNA molecule to the catalytic cleft.).The new chain tracing, based on new elements of electron density and a new interpretation that alters the assignment of specific amino acid residues to some of the original features<ref name="B" />. In the new model,the Inner arm is the extended chain motif (Met<sup>137</sup>to Ala<sup>142</sup>)is a segment of extended polypeptide chain that runs through the major groove of the DNA, roughly parallel to the DNA backbone<ref name="B" />. The outer arm is composed of two minor β strands linked together by a loop (the outer arm is 14 amino acids long, four of these amino acids belong to the loop).<br />
<br />
<br />
The specific recognition of ''Eco''RI of the GAATTC sequence is mediated by twelve hydrogen bonds (six bonds per subunit) originating from α helical recognition modules. Three amino acids are responsible for the recognition:Arg<sup>200</sup>, Glu<sup>144</sup> and Arg<sup>145</sup>. These aminoacids are shown in red <scene name='Sandbox_666/Grey_protein/3'>here</scene> .Each residue form two hydrogen bonds with Guanine and the adjacent Adenosine residues respectively. <br />
<br />
<br />
The reaction is due to a catalytic sequence motif which is found in most type II restriction endonucleases: the PD…(D/E)XK motif. For ''Eco''RI,<scene name='Sandbox_666/Catalytic_core/3'>this catalytic sequence</scene> is PD<sup>91</sup> …E<sup>111</sup>AK and the lysine residue is essential to the catalysis, but the proline residue is not important. This motif is also responsible for Mg2+ binding(Asp90 and Glu111).<ref>Structure and function of type II restriction endonucleases<br />
Alfred Pingoud, Albert Jeltsch<br />
Nucleic Acids Res. 2001 September 15; 29(18): 3705–3727.<br />
PMCID: PMC55916</ref><br />
<br />
<br />
There are several non-contiguous structural elements, which are involved in DNA recognition:<br />
* Four helix (two of each subunit) recognize the major groove and bring residues, whose interact with DNA bases and backbones<br />
* An extended peptide chain running through the major groove forms the specific contacts of the enzymes to the DNA.<br />
* One ß-strand parallel to the DNA backbone, which contains amino acid residues essential for catalysis (e.g.: residues engaged in phosphate contacts)<br />
* Two arms, which establish contacts with DNA backbones outside the recognition sequences.<br />
<br />
This binding by the major groove is due to the position of scissile phosphodiester bonds.<ref name="A">Recognition and cleavage of DNA by type-II restriction endonucleases, Pingoud A. & Jeltsch A. Eur.J.Biochem. 246,1-22 (1997)</ref><br />
<br />
[[Image:Catalytic 1ERI.png | thumb | The catalytic core of Eco RI with the target DNA|left|400px]]<br />
<br />
==Catalytic mechanism==<br />
<br />
<br />
The function of EcoRI is to cleave infectious DNA before it is methylated or before it begins to cause damage in the bacterial cell. That’s why EcoRI is able to bind non-specifically anywhere to the DNA and continuously scans the DNA in a linear diffusion process with an approximate rate of 7*106 bp.s-1. In this way, the protein targets very quickly its specific sequences and any recognition sites is forgotten. This linear diffusion is due to electrostatic interactions; thence the DNA is trapped by EcoRI but can move easily. Indeed during non-specific binding, there is no direct interaction with the bases of the DNA but there are only five interactions between amino acid residues from each subunit and the DNA phosphate groups. Moreover around 110 water molecules are located into this complex.<br />
<br />
It was observed that the enzyme makes pauses when the site is very similar to the recognition site (affinity of the enzyme to these sites). When one of these sites or a methylated site are cut, the reactional process is very slow and takes place only in one strand of the DNA, so the cellular DNA ligase can repair the nicked DNA. <br />
<br />
During the formation of the specific complex, the majority of the water molecules and ions are released and more and more direct interactions between the enzyme and the DNA are formed: some water molecules are immobilized between the enzyme and the DNA and formed hydrogen bounds, whose are important for the recognition and catalysis. Metal divalent ions, Mg2+, play also an essential role in the recognition process. The two homodimeres of EcoRI cooperate in the binding and cleavage of their substrate and are symmetrically bound to the DNA. The specific complex formation leads to conformational changes of the protein and the DNA, thus catalytic centers of the both subunits are activated and the both DNA strands are simultaneously cleaved. <br />
Specific binding bend the DNA by about 12° and involve interactions between EcoRI and DNA bases and phosphodiester backbones over approximately 10-12 bp. Moreover the residues involve to binding could participate at the catalysis. The number of interactions between EcoRI and the recognition sequence is maximal. <br />
The restriction enzymes are highly specific and highly cooperative.<ref name="A" /><br />
<br />
== Application of EcoRI in molecular biology ==<br />
<br />
<br />
Type II restriction endonuclease like ''Eco''RI are often used in molecular biology for their capacity to cut precisely DNA on specific restriction site. That makes them useful tools for gene cloning. By using two different restriction enzymes, it is possible to do directional cloning, which is very important if you want to insert a gene in an expression vector.<br />
<br />
== Links ==<br />
[http://www.rcsb.org/pdb/explore/explore.do?structureId=1ERI] 1ERI in the Protein Database (PDB)<br />
<br />
== References ==<br />
<references/> <br />
<br />
== Contributor ==<br />
<br />
Raphaël BILGER, Virginie GROSBOILLOT</div>Raphael Bilgerhttps://proteopedia.org/index.php?title=File:Image1_EcoRI.png&diff=1338227File:Image1 EcoRI.png2011-12-29T14:49:09Z<p>Raphael Bilger: The topology of the EcoRI type II restriction endonuclease</p>
<hr />
<div>The topology of the EcoRI type II restriction endonuclease</div>Raphael Bilgerhttps://proteopedia.org/index.php?title=Sandbox_666&diff=1338184Sandbox 6662011-12-29T14:10:17Z<p>Raphael Bilger: </p>
<hr />
<div>Hello this Sandbox is reserved for a student project. It describes the structure of the endonuclese restriction enzyme '''Eco RI'''.<br />
<br />
== Presentation ==<br />
<br />
<br />
''Eco''RI is a type II restriction endonuclease. It recognizes and cleaves DNA on a specific palindromic sequence: GAATTC. ''Eco''RI has been extracted from strain R ''Escherichia coli'', a common bacterium, which populates the intestine of mammalians. In bacteria, restriction enzymes protect the cell by cutting foreign DNA from bacteriophages (specific bacterial viruses) in both strands. Bacterial DNA is protected by a specific methylation of ''Eco''RI recognition sequences.<br />
<br />
== Reaction ==<br />
<br />
<br />
''Eco''RI (E.C. 3.1.21.4) is a hydrolase and its substrate is a double-strand DNA molecule and two water molecules. For its catalytic activity, ''Eco''RI needs a cofactor, which is the divalent ion Mg<sup>2+</sup>. ''Eco''RI hydrolyses the phosphodiester bond between the guanylic and adenylic residues resulting in 5’-phosphate sticky ends, which are complementary.<br />
<br />
== Structure ==<br />
<br />
{{STRUCTURE_1eri| PDB=1eri | SCENE= | size='500'}} <br />
''Eco''RI is composed of two homodimers, so it has two identical subunits (Representation of one <scene name='Sandbox_666/Monomer_structure/4'>subunit</scene>) of 31 kDa, but it is possible to have homotetramers at high concentrations. The constitutive monomers are 276 amino acids long. ''Eco''RI and all the other restriction enzymes show a common structural core, which is a α/β domain. The constitutive subunits of ''Eco''RI are organized into a single α/β domain (five strands <scene name='Sandbox_666/B_sheet/1'>β</scene> sheet, which is surrounded by <scene name='Sandbox_666/Helix/1'>two α helices</scene> ). Four of these five β strands are parallel whereas the fourth (β4) is in an anti-parallel orientation to the others.<ref>Refinement of ''Eco''RI endonuclease crystal structure: a revised protein chain tracing.<br />
Kim YC, Grable JC, Love R, Greene PJ, Rosenberg JM.</ref><br />
<br />
<br />
In the old model the N-terminal section of each subunit forms the inner arm which wraps around the DNA molecule (The arm brings the DNA molecule to the catalytic cleft.).The new chain tracing, based on new elements of electron density and a new interpretation that alters the assignment of specific amino acid residues to some of the original features<ref>Refinement of ''Eco''RI endonuclease crystal structure: a revised protein chain tracing.<br />
Kim YC, Grable JC, Love R, Greene PJ, Rosenberg JM.</ref>. In the new model,the Inner arm is the extended chain motif (Met<sup>137</sup>to Ala<sup>142</sup>)is a segment of extended polypeptide chain that runs through the major groove of the DNA, roughly parallel to the DNA backbone<ref>Refinement of ''Eco''RI endonuclease crystal structure: a revised protein chain tracing.<br />
Kim YC, Grable JC, Love R, Greene PJ, Rosenberg JM.</ref>. The outer arm is composed of two minor β strands linked together by a loop (the outer arm is 14 amino acids long, four of these amino acids belong to the loop).<br />
<br />
<br />
The specific recognition of ''Eco''RI of the GAATTC sequence is mediated by twelve hydrogen bonds (six bonds per subunit) originating from α helical recognition modules. Three amino acids are responsible for the recognition:Arg<sup>200</sup>, Glu<sup>144</sup> and Arg<sup>145</sup>. These aminoacids are shown in red <scene name='Sandbox_666/Grey_protein/3'>here</scene> .Each residue form two hydrogen bonds with Guanine and the adjacent Adenosine residues respectively. <br />
<br />
<br />
The reaction is due to a catalytic sequence motif which is found in most type II restriction endonucleases: the PD…(D/E)XK motif. For ''Eco''RI,<scene name='Sandbox_666/Catalytic_core/3'>this catalytic sequence</scene> is PD<sup>91</sup> …E<sup>111</sup>AK and the lysine residue is essential to the catalysis, but the proline residue is not important. This motif is also responsible for Mg2+ binding(Asp90 and Glu111).<ref>Structure and function of type II restriction endonucleases<br />
Alfred Pingoud, Albert Jeltsch<br />
Nucleic Acids Res. 2001 September 15; 29(18): 3705–3727.<br />
PMCID: PMC55916</ref><br />
<br />
There are several non-contiguous structural elements, which are involved in DNA recognition:<br />
<br />
-Four helix (two of each subunit) recognize the major groove and bring residues, whose interact with DNA bases and backbones<br />
<br />
-An extended peptide chain running through the major groove forms the specific contacts of the enzymes to the DNA.<br />
<br />
-One ß-strand parallel to the DNA backbone, which contains amino acid residues essential for catalysis (e.g.: residues engaged in phosphate contacts)<br />
<br />
-Two arms, which establish contacts with DNA backbones outside the recognition sequences.<br />
<br />
This binding by the major groove is due to the position of scissile phosphodiester bonds.<br />
<br />
[[Image:Catalytic 1ERI.png | thumb | The catalytic core of Eco RI with the target DNA|left|400px]]<br />
==Catalytic mechanism==<br />
<br />
<br />
The function of EcoRI is to cleave infectious DNA before it is methylated or before it begins to cause damage in the bacterial cell. That’s why EcoRI is able to bind non-specifically anywhere to the DNA and continuously scans the DNA in a linear diffusion process with an approximate rate of 7*106 bp.s-1. In this way, the protein targets very quickly its specific sequences and any recognition sites is forgotten. This linear diffusion is due to electrostatic interactions; thence the DNA is trapped by EcoRI but can move easily. Indeed during non-specific binding, there is no direct interaction with the bases of the DNA but there are only five interactions between amino acid residues from each subunit and the DNA phosphate groups. Moreover around 110 water molecules are located into this complex.<br />
<br />
It was observed that the enzyme makes pauses when the site is very similar to the recognition site (affinity of the enzyme to these sites). When one of these sites or a methylated site are cut, the reactional process is very slow and takes place only in one strand of the DNA, so the cellular DNA ligase can repair the nicked DNA. <br />
<br />
During the formation of the specific complex, the majority of the water molecules and ions are released and more and more direct interactions between the enzyme and the DNA are formed: some water molecules are immobilized between the enzyme and the DNA and formed hydrogen bounds, whose are important for the recognition and catalysis. Metal divalent ions, Mg2+, play also an essential role in the recognition process. The two homodimeres of EcoRI cooperate in the binding and cleavage of their substrate and are symmetrically bound to the DNA. The specific complex formation leads to conformational changes of the protein and the DNA, thus catalytic centers of the both subunits are activated and the both DNA strands are simultaneously cleaved. <br />
Specific binding bend the DNA by about 12° and involve interactions between EcoRI and DNA bases and phosphodiester backbones over approximately 10-12 bp. Moreover the residues involve to binding could participate at the catalysis. The number of interactions between EcoRI and the recognition sequence is maximal. <br />
The restriction enzymes are highly specific and highly cooperative.<ref>Recognition and cleavage of DNA by type-II restriction endonucleases, Pingoud A. & Jeltsch A. Eur.J.Biochem. 246,1-22 (1997)</ref><br />
<br />
== Application of EcoRI in molecular biology ==<br />
<br />
<br />
Type II restriction endonuclease like ''Eco''RI are often used in molecular biology for their capacity to cut precisely DNA on specific restriction site. That makes them useful tools for gene cloning. By using two different restriction enzymes, it is possible to do directional cloning, which is very important if you want to insert a gene in an expression vector.<br />
<br />
== Links ==<br />
[http://www.rcsb.org/pdb/explore/explore.do?structureId=1ERI] 1ERI in the Protein Database (PDB)<br />
<br />
== References ==<br />
<references/> <br />
<br />
== Contributor ==<br />
<br />
Raphaël BILGER Virginie GROSBOILLOT</div>Raphael Bilgerhttps://proteopedia.org/index.php?title=Sandbox_666&diff=1338144Sandbox 6662011-12-29T13:32:24Z<p>Raphael Bilger: </p>
<hr />
<div>Hello this Sandbox is reserved for a student project. It describes the structure of the endonuclese restriction enzyme '''Eco RI'''.<br />
<br />
== Presentation ==<br />
<br />
<br />
''Eco''RI is a type II restriction endonuclease. It recognizes and cleaves DNA on a specific palindromic sequence: GAATTC. ''Eco''RI has been extracted from strain R ''Escherichia coli'', a common bacterium, which populates the intestine of mammalians. In bacteria, restriction enzymes protect the cell by cutting foreign DNA from bacteriophages (specific bacterial viruses) in both strands. Bacterial DNA is protected by a specific methylation of ''Eco''RI recognition sequences.<br />
<br />
== Reaction ==<br />
<br />
<br />
''Eco''RI (E.C. 3.1.21.4) is a hydrolase and its substrate is a double-strand DNA molecule and two water molecules. For its catalytic activity, ''Eco''RI needs a cofactor, which is the divalent ion Mg<sup>2+</sup>. ''Eco''RI hydrolyses the phosphodiester bond between the guanylic and adenylic residues resulting in 5’-phosphate sticky ends, which are complementary.<br />
<br />
== Structure ==<br />
<br />
{{STRUCTURE_1eri| PDB=1eri | SCENE= | size='500'}} <br />
EcoRI is composed of two homodimers, so it has two identical subunits (Representation of one <scene name='Sandbox_666/Monomer_structure/4'>subunit</scene>) of 31 kDa, but it is possible to have homotetramers at high concentrations. The constitutive monomers are 276 amino acids long. EcoRI and all the other restriction enzymes show a common structural core, which is a α/β domain. The constitutive subunits of EcoRI are organized into a single α/β domain (five strands <scene name='Sandbox_666/B_sheet/1'>β</scene> sheet, which is surrounded by <scene name='Sandbox_666/Helix/1'>two α helices</scene> ). Four of these five β strands are parallel whereas the fourth (β4) is in an anti-parallel orientation to the others.<ref>Refinement of Eco RI endonuclease crystal structure: a revised protein chain tracing.<br />
Kim YC, Grable JC, Love R, Greene PJ, Rosenberg JM.</ref><br />
<br />
<br />
In the old model the N-terminal section of each subunit forms the inner arm which wraps around the DNA molecule (The arm brings the DNA molecule to the catalytic cleft.).The new chain tracing, based on new elements of electron density and a new interpretation that alters the assignment of specific amino acid residues to some of the original features<ref>Refinement of Eco RI endonuclease crystal structure: a revised protein chain tracing.<br />
Kim YC, Grable JC, Love R, Greene PJ, Rosenberg JM.</ref>. In the new model,the Inner arm is the extended chain motif (Met<sup>137</sup>to Ala<sup>142</sup>)is a segment of extended polypeptide chain that runs through the major groove of the DNA, roughly parallel to the DNA backbone<ref>Refinement of Eco RI endonuclease crystal structure: a revised protein chain tracing.<br />
Kim YC, Grable JC, Love R, Greene PJ, Rosenberg JM.</ref>. The Outer arm is composed of two minor β strands linked together by a loop (the outer arm is 14 amino acids long, four of these amino acids belong to the loop).<br />
<br />
<br />
The specific recognition of EcoRI of the GAATTC sequence is mediated by twelve hydrogen bonds (six bonds per subunit) originating from α helical recognition modules. Three amino acids are responsible for the recognition:Arg<sup>200</sup>, Glu<sup>144</sup> and Arg<sup>145</sup>. These aminoacids are shown in red <scene name='Sandbox_666/Grey_protein/3'>here</scene> .Each residue form two hydrogen bonds with Guanine and the adjacent Adenosine residues respectively. <br />
<br />
<br />
The reaction is due to a catalytic sequence motif which is found in most type II restriction endonucleases: the PD…D/EXK motif. For EcoRI,<scene name='Sandbox_666/Catalytic_core/3'>this catalytic sequence</scene> is PD<sup>91</sup> …E<sup>111</sup>AK. This motif is also responsible for Mg2+ binding(Asp90 and Glu111).<br />
[[Image:Catalytic 1ERI.png | thumb | The catalytic core of Eco RI with the target DNA|left|400px]]<ref><br />
Structure and function of type II restriction endonucleases<br />
Alfred Pingoud, Albert Jeltsch<br />
Nucleic Acids Res. 2001 September 15; 29(18): 3705–3727.<br />
PMCID: PMC55916</ref><br />
<br />
== Catalytic Mechanism ==<br />
The function of EcoRI is to cleave infectious DNA before it is methylated or before it begins to cause damage in the bacterial cell. That’s why EcoRI is able to bind non-specifically anywhere to the DNA and continuously scans the DNA in a linear diffusion process with an approximate rate of 7*106 bp.s-1. In this way, the protein targets very quickly its specific sequences and any recognition sites is forgotten. This linear diffusion is due to electrostatic interactions; thence the DNA is trapped by EcoRI but can move easily. Indeed during non-specific binding, there is no direct interaction with the bases of the DNA but there are only five interactions between amino acid residues from each subunit and the DNA phosphate groups. Moreover around 110 water molecules are located into this complex.<br />
<br />
It was observed that the enzyme makes pauses when the site is very similar to the recognition site (affinity of the enzyme to these sites). When one of these sites or a methylated site are cut, the reactional process is very slow and takes place only in one strand of the DNA, so the cellular DNA ligase can repair the nicked DNA. <br />
<br />
During the formation of the specific complex, the majority of the water molecules and ions are released and more and more direct interactions between the enzyme and the DNA are formed: some water molecules are immobilized between the enzyme and the DNA and formed hydrogen bounds, whose are important for the recognition and catalysis. Metal divalent ions, Mg2+, play also an essential role in the recognition process. The two homodimeres of EcoRI cooperate in the binding and cleavage of their substrate and are symmetrically bound to the DNA. The specific complex formation leads to conformational changes of the protein and the DNA, thus catalytic centers of the both subunits are activated and the both DNA strands are simultaneously cleaved. <br />
Specific binding bend the DNA by about 12° and involve interactions between EcoRI and DNA bases and phosphodiester backbones over approximately 10-12 bp. Moreover the residues involve to binding could participate at the catalysis. The number of interactions between EcoRI and the recognition sequence is maximal. <br />
The restriction enzymes are highly specific and highly cooperative.<br />
<br />
== Application of EcoRI in molecular biology ==<br />
<br />
<br />
Type II restriction endonuclease like EcoRI are often used in molecular biology for their capacity to cut precisely DNA on specific restriction site. That makes them useful tools for gene cloning. By using two different restriction enzymes, it is possible to do directional cloning, which is very important if you want to insert a gene in an expression vector.<br />
<br />
== Links ==<br />
[http://www.rcsb.org/pdb/explore/explore.do?structureId=1ERI] 1ERI in the Protein Database (PDB)<br />
<br />
== References ==<br />
<references/> 6. ↑ Recognition and cleavage of DNA by type-II restriction endonucleases, Pingoud A. & Jeltsch A. Eur.J.Biochem. 246,1-22 (1997)<br />
<br />
== Contributor ==<br />
<br />
Raphaël BILGER Virginie GROSBOILLOT</div>Raphael Bilgerhttps://proteopedia.org/index.php?title=Sandbox_666&diff=1338100Sandbox 6662011-12-29T11:26:08Z<p>Raphael Bilger: </p>
<hr />
<div>Hello this Sandbox is reserved for a student project. It describes the structure of the endonuclese restriction enzyme '''Eco RI'''.<br />
{{STRUCTURE_1eri| PDB=1eri | SCENE= | size='500'}} <br />
== Presentation ==<br />
<br />
<br />
EcoRI is a type II restriction endonuclease. It recognizes and cleaves DNA on a specific palindromic sequence: GAATTC. EcoRI has been extracted from strain R Escherichia coli, a common bacterium which populates the intestine of mammalians. In bacteria, restriction enzymes protect the cell by cutting foreign DNA from bacteriophages (specific bacterial viruses).<br />
<br />
== Reaction ==<br />
<br />
<br />
EcoRI (E.C. 3.1.21.4) is a hydrolase and its substrate is a double-strand DNA molecule and two water molecules. For its catalytic activity, EcoRI needs a cofactor which is the divalent ion Mg<sup>2+</sup>. EcoRI hydrolyses the phosphodiester bond between the guanylic and adenylic residues resulting in 5’-phosphate sticky ends which are complementary.<br />
<br />
== Structure ==<br />
<br />
<br />
EcoRI is a homodimer, so it has two identical subunits (Representation of one <scene name='Sandbox_666/Monomer_structure/4'>subunit</scene>) of 31 kDa, but it possible to have homotetramers at high concentrations. The constitutive monomers are 276 amino acids long. EcoRI and all the other restriction enzymes show a common structural core which is a α/β domain. The constitutive subunits of EcoRI are organized into a single α/β domain (five stranded <scene name='Sandbox_666/B_sheet/1'>β</scene> sheet which is surrounded by <scene name='Sandbox_666/Helix/1'>α helices</scene> ). Four of these five β strands are parallel whereas the fourth (β4) is in an anti-parallel orientation to the others.<ref>Refinement of Eco RI endonuclease crystal structure: a revised protein chain tracing.<br />
Kim YC, Grable JC, Love R, Greene PJ, Rosenberg JM.</ref><br />
<br />
<br />
In the old model the N-terminal section of each subunit forms the inner arm which wraps around the DNA molecule (The arm brings the DNA molecule to the catalytic cleft.).The new chain tracing, based on new elements of electron density and anew interpretation that alters the assignment of specific amino acid residues to some of the original features<ref>Refinement of Eco RI endonuclease crystal structure: a revised protein chain tracing.<br />
Kim YC, Grable JC, Love R, Greene PJ, Rosenberg JM.</ref>. In the new model,the Inner arm is the extended chain motif (Met<sup>137</sup>to Ala<sup>142</sup>)is a segment of extended polypeptide chain that runs through the major groove of the DNA, roughly parallel to the DNA backbone<ref>Refinement of Eco RI endonuclease crystal structure: a revised protein chain tracing.<br />
Kim YC, Grable JC, Love R, Greene PJ, Rosenberg JM.</ref>. The Outer arm is composed of two minor β strands linked together by a loop (the outer arm is 14 amino acids long, four of these amino acids belong to the loop).<br />
<br />
<br />
The specific recognition of EcoRI of the GAATTC sequence is mediated by twelve hydrogen bonds (six bonds per subunit) originating from α helical recognition modules. Three amino acids are responsible for the recognition:Arg<sup>200</sup>, Glu<sup>144</sup> and Arg<sup>145</sup>. These aminoacids are shown in red <scene name='Sandbox_666/Grey_protein/3'>here</scene> .Each residue form two hydrogen bonds with Guanine and the adjacent Adenosine residues respectively. <br />
<br />
<br />
The reaction is due to a catalytic sequence motif which is found in most type II restriction endonucleases: the PD…D/EXK motif. For EcoRI,<scene name='Sandbox_666/Catalytic_core/3'>this catalytic sequence</scene> is PD<sup>91</sup> …E<sup>111</sup>AK. This motif is also responsible for Mg2+ binding(Asp90 and Glu111).<br />
[[Image:Catalytic 1ERI.png | thumb | The catalytic core of Eco RI with the target DNA|left|400px]]<ref><br />
Structure and function of type II restriction endonucleases<br />
Alfred Pingoud, Albert Jeltsch<br />
Nucleic Acids Res. 2001 September 15; 29(18): 3705–3727.<br />
PMCID: PMC55916</ref><br />
<br />
== Catalytic Mechanism ==<br />
The function of EcoRI is to cleave infectious DNA before it is methylated or before it begins to cause damage in the bacterial cell. That’s why EcoRI is able to bind non-specifically anywhere to the DNA and continuously scans the DNA in a linear diffusion process with an approximate rate of 7*106 bp.s-1. In this way, the protein targets very quickly its specific sequences and any recognition sites is forgotten. This linear diffusion is due to electrostatic interactions; thence the DNA is trapped by EcoRI but can move easily. Indeed during non-specific binding, there is no direct interaction with the bases of the DNA but there are only five interactions between amino acid residues from each subunit and the DNA phosphate groups. Moreover around 110 water molecules are located into this complex.<br />
<br />
It was observed that the enzyme makes pauses when the site is very similar to the recognition site (affinity of the enzyme to these sites). When one of these sites or a methylated site are cut, the reactional process is very slow and takes place only in one strand of the DNA, so the cellular DNA ligase can repair the nicked DNA. <br />
<br />
During the formation of the specific complex, the majority of the water molecules and ions are released and more and more direct interactions between the enzyme and the DNA are formed: some water molecules are immobilized between the enzyme and the DNA and formed hydrogen bounds, whose are important for the recognition and catalysis. Metal divalent ions, Mg2+, play also an essential role in the recognition process. The two homodimeres of EcoRI cooperate in the binding and cleavage of their substrate and are symmetrically bound to the DNA. The specific complex formation leads to conformational changes of the protein and the DNA, thus catalytic centers of the both subunits are activated and the both DNA strands are simultaneously cleaved. <br />
Specific binding bend the DNA by about 12° and involve interactions between EcoRI and DNA bases and phosphodiester backbones over approximately 10-12 bp. Moreover the residues involve to binding could participate at the catalysis. The number of interactions between EcoRI and the recognition sequence is maximal. <br />
The restriction enzymes are highly specific and highly cooperative.<br />
<br />
== Application of EcoRI in molecular biology ==<br />
<br />
<br />
Type II restriction endonuclease like EcoRI are often used in molecular biology for their capacity to cut precisely DNA on specific restriction site. That makes them useful tools for gene cloning. By using two different restriction enzymes, it is possible to do directional cloning which is very important if you want to insert a gene in an expression vector.<br />
<br />
== Links ==<br />
[http://www.rcsb.org/pdb/explore/explore.do?structureId=1ERI] 1ERI in the Protein Database (PDB)<br />
<br />
== References ==<br />
<references/><br />
<br />
<br />
== Contributor ==<br />
<br />
Raphaël BILGER Virginie GROSBOILLOT</div>Raphael Bilgerhttps://proteopedia.org/index.php?title=Sandbox_666&diff=1338056Sandbox 6662011-12-29T08:49:51Z<p>Raphael Bilger: </p>
<hr />
<div>Hello this Sandbox is reserved for a student project. It describes the structure of the endonuclese restriction enzyme '''Eco RI'''.<br />
{{STRUCTURE_1eri| PDB=1eri | SCENE= | size='500'}} <br />
== Presentation ==<br />
<br />
<br />
EcoRI is a type II restriction endonuclease. It recognizes and cleaves DNA on a specific palindromic sequence: GAATTC. EcoRI has been extracted from strain R Escherichia coli, a common bacterium which populates the intestine of mammalians. In bacteria, restriction enzymes protect the cell by cutting foreign DNA from bacteriophages (specific bacterial viruses).<br />
<br />
== Reaction ==<br />
<br />
<br />
EcoRI (E.C. 3.1.21.4) is a hydrolase and its substrate is a double-strand DNA molecule and two water molecules. For its catalytic activity, EcoRI needs a cofactor which is the divalent ion Mg<sup>2+</sup>. EcoRI hydrolyses the phosphodiester bond between the guanylic and adenylic residues resulting in 5’-phosphate sticky ends which are complementary.<br />
<br />
== Structure ==<br />
<br />
<br />
EcoRI is a homodimer, so it has two identical subunits (Representation of one <scene name='Sandbox_666/Monomer_structure/4'>subunit</scene>) of 31 kDa, but it possible to have homotetramers at high concentrations. The constitutive monomers are 276 amino acids long. EcoRI and all the other restriction enzymes show a common structural core which is a α/β domain. The constitutive subunits of EcoRI are organized into a single α/β domain (five stranded <scene name='Sandbox_666/B_sheet/1'>β</scene> sheet which is surrounded by <scene name='Sandbox_666/Helix/1'>α helices</scene> ). Four of these five β strands are parallel whereas the fourth (β4) is in an anti-parallel orientation to the others.<ref>Refinement of Eco RI endonuclease crystal structure: a revised protein chain tracing.<br />
Kim YC, Grable JC, Love R, Greene PJ, Rosenberg JM.</ref><br />
<br />
<br />
In the old model the N-terminal section of each subunit forms the inner arm which wraps around the DNA molecule (The arm brings the DNA molecule to the catalytic cleft.).The new chain tracing, based on new elements of electron density and anew interpretation that alters the assignment of specific amino acid residues to some of the original features<ref>Refinement of Eco RI endonuclease crystal structure: a revised protein chain tracing.<br />
Kim YC, Grable JC, Love R, Greene PJ, Rosenberg JM.</ref>. In the new model,the Inner arm is the extended chain motif (Met<sup>137</sup>to Ala<sup>142</sup>)is a segment of extended polypeptide chain that runs through the major groove of the DNA, roughly parallel to the DNA backbone<ref>Refinement of Eco RI endonuclease crystal structure: a revised protein chain tracing.<br />
Kim YC, Grable JC, Love R, Greene PJ, Rosenberg JM.</ref>. The Outer arm is composed of two minor β strands linked together by a loop (the outer arm is 14 amino acids long, four of these amino acids belong to the loop).<br />
<br />
<br />
The specific recognition of EcoRI of the GAATTC sequence is mediated by twelve hydrogen bonds (six bonds per subunit) originating from α helical recognition modules. Three amino acids are responsible for the recognition:Arg<sup>200</sup>, Glu<sup>144</sup> and Arg<sup>145</sup>. These aminoacids are shown in red <scene name='Sandbox_666/Grey_protein/3'>here</scene> .Each residue form two hydrogen bonds with Guanine and the adjacent Adenosine residues respectively. <br />
<br />
<br />
The reaction is due to a catalytic sequence motif which is found in most type II restriction endonucleases: the PD…D/EXK motif. For EcoRI,<scene name='Sandbox_666/Catalytic_core/3'>this catalytic sequence</scene> is PD<sup>91</sup> …E<sup>111</sup>AK. This motif is also responsible for Mg2+ binding(Asp90 and Glu111).<br />
[[Image:Catalytic 1ERI.png | thumb | The catalytic core of Eco RI with the target DNA|left|400px]]<ref><br />
Structure and function of type II restriction endonucleases<br />
Alfred Pingoud, Albert Jeltsch<br />
Nucleic Acids Res. 2001 September 15; 29(18): 3705–3727.<br />
PMCID: PMC55916</ref><br />
<br />
<br />
== Application of EcoRI in molecular biology ==<br />
<br />
<br />
Type II restriction endonuclease like EcoRI are often used in molecular biology for their capacity to cut precisely DNA on specific restriction site. That makes them useful tools for gene cloning. By using two different restriction enzymes, it is possible to do directional cloning which is very important if you want to insert a gene in an expression vector.<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
== Links ==<br />
[http://www.rcsb.org/pdb/explore/explore.do?structureId=1ERI] 1ERI in the Protein Database (PDB)<br />
<br />
== References ==<br />
<references/><br />
<br />
<br />
== Contributor ==<br />
<br />
Raphaël BILGER Virginie GROSBOILLOT</div>Raphael Bilgerhttps://proteopedia.org/index.php?title=Sandbox_666&diff=1337995Sandbox 6662011-12-28T16:34:21Z<p>Raphael Bilger: </p>
<hr />
<div>Hello this Sandbox is reserved for a student project. It describes the structure of the endonuclese restriction enzyme '''Eco RI'''.<br />
{{STRUCTURE_1eri| PDB=1eri | SCENE= | size='500'}} <br />
== Presentation ==<br />
<br />
<br />
EcoRI is a type II restriction endonuclease. It recognizes and cleaves DNA on a specific palindromic sequence: GAATTC. EcoRI has been extracted from strain R Escherichia coli, a common bacterium which populates the intestine of mammalians. In bacteria, restriction enzymes protect the cell by cutting foreign DNA from bacteriophages (specific bacterial viruses).<br />
<br />
== Reaction ==<br />
<br />
<br />
EcoRI (E.C. 3.1.21.4) is a hydrolase and its substrate is a double-strand DNA molecule and two water molecules. For its catalytic activity, EcoRI needs a cofactor which is the divalent ion Mg<sup>2+</sup>. EcoRI hydrolyses the phosphodiester bond between the guanylic and adenylic residues resulting in 5’-phosphate sticky ends which are complementary.<br />
<br />
== Structure ==<br />
<br />
<br />
EcoRI is a homodimer, so it has two identical subunits (Representation of one <scene name='Sandbox_666/Monomer_structure/4'>subunit</scene>) of 31 kDa, but it possible to have homotetramers at high concentrations. The constitutive monomers are 276 amino acids long. EcoRI and all the other restriction enzymes show a common structural core which is a α/β domain. The constitutive subunits of EcoRI are organized into a single α/β domain (five stranded <scene name='Sandbox_666/B_sheet/1'>β</scene> sheet which is surrounded by <scene name='Sandbox_666/Helix/1'>α helices</scene> ). Four of these five β strands are parallel whereas the fourth (β4) is in an anti-parallel orientation to the others.<ref>Refinement of Eco RI endonuclease crystal structure: a revised protein chain tracing.<br />
Kim YC, Grable JC, Love R, Greene PJ, Rosenberg JM.</ref><br />
<br />
<br />
In the old model the N-terminal section of each subunit forms the inner arm which wraps around the DNA molecule (The arm brings the DNA molecule to the catalytic cleft.).The new chain tracing, based on new elements of electron density and anew interpretation that alters the assignment of specific amino acid residues to some of the original features<ref>Refinement of Eco RI endonuclease crystal structure: a revised protein chain tracing.<br />
Kim YC, Grable JC, Love R, Greene PJ, Rosenberg JM.</ref>. In the new model,the Inner arm is the extended chain motif (Met<sup>137</sup>to Ala<sup>142</sup>)is a segment of extended polypeptide chain that runs through the major groove of the DNA, roughly parallel to the DNA backbone<ref>Refinement of Eco RI endonuclease crystal structure: a revised protein chain tracing.<br />
Kim YC, Grable JC, Love R, Greene PJ, Rosenberg JM.</ref>. The Outer arm is composed of two minor β strands linked together by a loop (the outer arm is 14 amino acids long, four of these amino acids belong to the loop).<br />
<br />
<br />
The specific recognition of EcoRI of the GAATTC sequence is mediated by twelve hydrogen bonds (six bonds per subunit) originating from α helical recognition modules. Three amino acids are responsible for the recognition:Arg<sup>200</sup>, Glu<sup>144</sup> and Arg<sup>145</sup>. These aminoacids are shown in red <scene name='Sandbox_666/Grey_protein/3'>here</scene> .Each residue form two hydrogen bonds with Guanine and the adjacent Adenosine residues respectively. <br />
<br />
<br />
The reaction is due to a catalytic sequence motif which is found in most type II restriction endonucleases: the PD…D/EXK motif. For EcoRI,<scene name='Sandbox_666/Catalytic_core/3'>this catalytic sequence</scene> is PD<sup>91</sup> …E<sup>111</sup>AK. This motif is also responsible for Mg2+ binding(Asp90 and Glu111).<br />
[[Image:Catalytic 1ERI.png | thumb | The catalytic core of Eco RI with the target DNA|left|400px]]<ref><br />
Structure and function of type II restriction endonucleases<br />
Alfred Pingoud, Albert Jeltsch<br />
Nucleic Acids Res. 2001 September 15; 29(18): 3705–3727.<br />
PMCID: PMC55916</ref><br />
<br />
<br />
== Application of EcoRI in molecular biology ==<br />
<br />
<br />
Type II restriction endonuclease like EcoRI are often used in molecular biology for their capacity to cut precisely DNA on specific restriction site. That makes them useful tools for gene cloning. By using two different restriction enzymes, it is possible to do directional cloning which is very important if you want to insert a gene in an expression vector.<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
== Links ==<br />
[http://www.rcsb.org/pdb/explore/explore.do?structureId=1ERI] 1ERI in the Protein Database (PDB)<br />
<br />
== References ==<br />
<references/><br />
<br />
<br />
== Contributor ==<br />
<br />
Raphaël BILGER</div>Raphael Bilgerhttps://proteopedia.org/index.php?title=Sandbox_666&diff=1337994Sandbox 6662011-12-28T16:29:37Z<p>Raphael Bilger: </p>
<hr />
<div>Hello this Sandbox is reserved for a student project. It describes the structure of the endonuclese restriction enzyme '''Eco RI'''.<br />
{{STRUCTURE_1eri| PDB=1eri | SCENE= | size='500'}} <br />
== Presentation ==<br />
<br />
<br />
EcoRI is a type II restriction endonuclease. It recognizes and cleaves DNA on a specific palindromic sequence: GAATTC. EcoRI has been extracted from strain R Escherichia coli, a common bacterium which populates the intestine of mammalians. In bacteria, restriction enzymes protect the cell by cutting foreign DNA from bacteriophages (specific bacterial viruses).<br />
<br />
== Reaction ==<br />
<br />
<br />
EcoRI (E.C. 3.1.21.4) is a hydrolase and its substrate is a double-strand DNA molecule and two water molecules. For its catalytic activity, EcoRI needs a cofactor which is the divalent ion Mg<sup>2+</sup>. EcoRI hydrolyses the phosphodiester bond between the guanylic and adenylic residues resulting in 5’-phosphate sticky ends which are complementary.<br />
<br />
== Structure ==<br />
<br />
<br />
EcoRI is a homodimer, so it has two identical subunits (Representation of one <scene name='Sandbox_666/Monomer_structure/4'>subunit</scene>) of 31 kDa, but it possible to have homotetramers at high concentrations. The constitutive monomers are 276 amino acids long. EcoRI and all the other restriction enzymes show a common structural core which is a α/β domain. The constitutive subunits of EcoRI are organized into a single α/β domain (five stranded <scene name='Sandbox_666/B_sheet/1'>β</scene> sheet which is surrounded by <scene name='Sandbox_666/Helix/1'>α helices</scene> ). Four of these five β strands are parallel whereas the fourth (β4) is in an anti-parallel orientation to the others.<ref>Refinement of Eco RI endonuclease crystal structure: a revised protein chain tracing.<br />
Kim YC, Grable JC, Love R, Greene PJ, Rosenberg JM.</ref><br />
<br />
<br />
In the old model the N-terminal section of each subunit forms the inner arm which wraps around the DNA molecule (The arm brings the DNA molecule to the catalytic cleft.).The new chain tracing, based on new elements of electron density and anew interpretation that alters the assignment of specific amino acid residues to some of the original features<ref>Refinement of Eco RI endonuclease crystal structure: a revised protein chain tracing.<br />
Kim YC, Grable JC, Love R, Greene PJ, Rosenberg JM.</ref>. In the new model,the Inner arm is the extended chain motif (Met<sup>137</sup>to Ala<sup>142</sup>)is a segment of extended polypeptide chain that runs through the major groove of the DNA, roughly parallel to the DNA backbone<ref>Refinement of Eco RI endonuclease crystal structure: a revised protein chain tracing.<br />
Kim YC, Grable JC, Love R, Greene PJ, Rosenberg JM.</ref>. The Outer arm is composed of two minor β strands linked together by a loop (the outer arm is 14 amino acids long, four of these amino acids belong to the loop).<br />
<br />
<br />
The specific recognition of EcoRI of the GAATTC sequence is mediated by twelve hydrogen bonds (six bonds per subunit) originating from α helical recognition modules. Three amino acids are responsible for the recognition:Arg<sup>200</sup>, Glu<sup>144</sup> and Arg<sup>145</sup>. These aminoacids are shown in red <scene name='Sandbox_666/Grey_protein/3'>here</scene> .Each residue form two hydrogen bonds with Guanine and the adjacent Adenosine residues respectively. <br />
<br />
<br />
The reaction is due to a catalytic sequence motif which is found in most type II restriction endonucleases: the PD…D/EXK motif. For EcoRI,<scene name='Sandbox_666/Catalytic_core/3'>this catalytic sequence</scene> is PD<sup>91</sup> …E<sup>111</sup>AK. This motif is also responsible for Mg2+ binding(Asp90 and Glu111). [[Image:Catalytic 1ERI.png | thumb | The catalytic core of Eco RI with the target DNA|left|500px]]<ref><br />
Structure and function of type II restriction endonucleases<br />
Alfred Pingoud, Albert Jeltsch<br />
Nucleic Acids Res. 2001 September 15; 29(18): 3705–3727.<br />
PMCID: PMC55916</ref><br />
<br />
<br />
== Application of EcoRI in molecular biology ==<br />
<br />
<br />
Type II restriction endonuclease like EcoRI are often used in molecular biology for their capacity to cut precisely DNA on specific restriction site. That makes them useful tools for gene cloning. By using two different restriction enzymes, it is possible to do directional cloning which is very important if you want to insert a gene in an expression vector.<br />
<br />
== Links ==<br />
[http://www.rcsb.org/pdb/explore/explore.do?structureId=1ERI] 1ERI in the Protein Database (PDB)<br />
<br />
== References ==<br />
<references/><br />
<br />
<br />
== Contributor ==<br />
<br />
Raphaël BILGER</div>Raphael Bilgerhttps://proteopedia.org/index.php?title=Sandbox_666&diff=1334073Sandbox 6662011-12-27T16:22:17Z<p>Raphael Bilger: </p>
<hr />
<div>Hello this Sandbox is reserved for a student project. It describes the structure of the endonuclese restriction enzyme '''Eco RI'''.<br />
{{STRUCTURE_1eri| PDB=1eri | SCENE= | size='500'}} <br />
== Presentation ==<br />
<br />
<br />
EcoRI is a type II restriction endonuclease. It recognizes and cleaves DNA on a specific palindromic sequence: GAATTC. EcoRI has been extracted from strain R Escherichia coli, a common bacterium which populates the intestine of mammalians. In bacteria, restriction enzymes protect the cell by cutting foreign DNA from bacteriophages (specific bacterial viruses).<br />
<br />
== Reaction ==<br />
<br />
<br />
EcoRI (E.C. 3.1.21.4) is a hydrolase and its substrate is a double-strand DNA molecule and two water molecules. For its catalytic activity, EcoRI needs a cofactor which is the divalent ion Mg<sup>2+</sup>. EcoRI hydrolyses the phosphodiester bond between the guanylic and adenylic residues resulting in 5’-phosphate sticky ends which are complementary.<br />
<br />
== Structure ==<br />
<br />
<br />
EcoRI is a homodimer, so it has two identical subunits (Representation of one <scene name='Sandbox_666/Monomer_structure/4'>subunit</scene>) of 31 kDa, but it possible to have homotetramers at high concentrations. The constitutive monomers are 276 amino acids long. EcoRI and all the other restriction enzymes show a common structural core which is a α/β domain. The constitutive subunits of EcoRI are organized into a single α/β domain (five stranded <scene name='Sandbox_666/B_sheet/1'>β</scene> sheet which is surrounded by <scene name='Sandbox_666/Helix/1'>α helices</scene> ). Four of these five β strands are parallel whereas the fourth (β4) is in an anti-parallel orientation to the others.<ref>Refinement of Eco RI endonuclease crystal structure: a revised protein chain tracing.<br />
Kim YC, Grable JC, Love R, Greene PJ, Rosenberg JM.</ref><br />
<br />
<br />
In the old model the N-terminal section of each subunit forms the inner arm which wraps around the DNA molecule (The arm brings the DNA molecule to the catalytic cleft.).The new chain tracing, based on new elements of electron density and anew interpretation that alters the assignment of specific amino acid residues to some of the original features<ref>Refinement of Eco RI endonuclease crystal structure: a revised protein chain tracing.<br />
Kim YC, Grable JC, Love R, Greene PJ, Rosenberg JM.</ref>. In the new model,the Inner arm is the extended chain motif (Met<sup>137</sup>to Ala<sup>142</sup>)is a segment of extended polypeptide chain that runs through the major groove of the DNA, roughly parallel to the DNA backbone<ref>Refinement of Eco RI endonuclease crystal structure: a revised protein chain tracing.<br />
Kim YC, Grable JC, Love R, Greene PJ, Rosenberg JM.</ref>. The Outer arm is composed of two minor β strands linked together by a loop (the outer arm is 14 amino acids long, four of these amino acids belong to the loop).<br />
<br />
<br />
The specific recognition of EcoRI of the GAATTC sequence is mediated by twelve hydrogen bonds (six bonds per subunit) originating from α helical recognition modules. Three amino acids are responsible for the recognition:Arg<sup>200</sup>, Glu<sup>144</sup> and Arg<sup>145</sup>. These aminoacids are shown in red <scene name='Sandbox_666/Grey_protein/3'>here</scene> .Each residue form two hydrogen bonds with Guanine and the adjacent Adenosine residues respectively. <br />
<br />
<br />
The reaction is due to a catalytic sequence motif which is found in most type II restriction endonucleases: the PD…D/EXK motif. For EcoRI,<scene name='Sandbox_666/Catalytic_core/3'>this catalytic sequence</scene> is PD<sup>91</sup> …E<sup>111</sup>AK. This motif is also responsible for Mg2+ binding(Asp90 and Glu111). [[Image:Catalytic 1ERI.png | thumb | The catalytic core of Eco RI with the target DNA]]<ref><br />
Structure and function of type II restriction endonucleases<br />
Alfred Pingoud, Albert Jeltsch<br />
Nucleic Acids Res. 2001 September 15; 29(18): 3705–3727.<br />
PMCID: PMC55916</ref><br />
<br />
<br />
== Application of EcoRI in molecular biology ==<br />
<br />
<br />
Type II restriction endonuclease like EcoRI are often used in molecular biology for their capacity to cut precisely DNA on specific restriction site. That makes them useful tools for gene cloning. By using two different restriction enzymes, it is possible to do directional cloning which is very important if you want to insert a gene in an expression vector.<br />
<br />
== Links ==<br />
[http://www.rcsb.org/pdb/explore/explore.do?structureId=1ERI] 1ERI in the Protein Database (PDB)<br />
<br />
== References ==<br />
<references/><br />
<br />
<br />
== Contributor ==<br />
<br />
Raphaël BILGER</div>Raphael Bilgerhttps://proteopedia.org/index.php?title=Sandbox_666&diff=1334072Sandbox 6662011-12-27T16:20:06Z<p>Raphael Bilger: </p>
<hr />
<div>Hello this Sandbox is reserved for a student project. It describes the structure of the endonuclese restriction enzyme Eco RI.<br />
{{STRUCTURE_1eri| PDB=1eri | SCENE= | size='500'}} <br />
== Presentation ==<br />
<br />
<br />
EcoRI is a type II restriction endonuclease. It recognizes and cleaves DNA on a specific palindromic sequence: GAATTC. EcoRI has been extracted from strain R Escherichia coli, a common bacterium which populates the intestine of mammalians. In bacteria, restriction enzymes protect the cell by cutting foreign DNA from bacteriophages (specific bacterial viruses).<br />
<br />
== Reaction ==<br />
<br />
<br />
EcoRI (E.C. 3.1.21.4) is a hydrolase and its substrate is a double-strand DNA molecule and two water molecules. For its catalytic activity, EcoRI needs a cofactor which is the divalent ion Mg<sup>2+</sup>. EcoRI hydrolyses the phosphodiester bond between the guanylic and adenylic residues resulting in 5’-phosphate sticky ends which are complementary.<br />
<br />
== Structure ==<br />
<br />
<br />
EcoRI is a homodimer, so it has two identical subunits (Representation of one <scene name='Sandbox_666/Monomer_structure/4'>subunit</scene>) of 31 kDa, but it possible to have homotetramers at high concentrations. The constitutive monomers are 276 amino acids long. EcoRI and all the other restriction enzymes show a common structural core which is a α/β domain. The constitutive subunits of EcoRI are organized into a single α/β domain (five stranded <scene name='Sandbox_666/B_sheet/1'>β</scene> sheet which is surrounded by <scene name='Sandbox_666/Helix/1'>α helices</scene> ). Four of these five β strands are parallel whereas the fourth (β4) is in an anti-parallel orientation to the others.<ref>Refinement of Eco RI endonuclease crystal structure: a revised protein chain tracing.<br />
Kim YC, Grable JC, Love R, Greene PJ, Rosenberg JM.</ref><br />
<br />
<br />
In the old model the N-terminal section of each subunit forms the inner arm which wraps around the DNA molecule (The arm brings the DNA molecule to the catalytic cleft.).The new chain tracing, based on new elements of electron density and anew interpretation that alters the assignment of specific amino acid residues to some of the original features<ref>Refinement of Eco RI endonuclease crystal structure: a revised protein chain tracing.<br />
Kim YC, Grable JC, Love R, Greene PJ, Rosenberg JM.</ref>. In the new model,the Inner arm is the extended chain motif (Met<sup>137</sup>to Ala<sup>142</sup>)is a segment of extended polypeptide chain that runs through the major groove of the DNA, roughly parallel to the DNA backbone<ref>Refinement of Eco RI endonuclease crystal structure: a revised protein chain tracing.<br />
Kim YC, Grable JC, Love R, Greene PJ, Rosenberg JM.</ref>. The Outer arm is composed of two minor β strands linked together by a loop (the outer arm is 14 amino acids long, four of these amino acids belong to the loop).<br />
<br />
<br />
The specific recognition of EcoRI of the GAATTC sequence is mediated by twelve hydrogen bonds (six bonds per subunit) originating from α helical recognition modules. Three amino acids are responsible for the recognition:Arg<sup>200</sup>, Glu<sup>144</sup> and Arg<sup>145</sup>. These aminoacids are shown in red <scene name='Sandbox_666/Grey_protein/3'>here</scene> .Each residue form two hydrogen bonds with Guanine and the adjacent Adenosine residues respectively. <br />
<br />
<br />
The reaction is due to a catalytic sequence motif which is found in most type II restriction endonucleases: the PD…D/EXK motif. For EcoRI,<scene name='Sandbox_666/Catalytic_core/3'>this catalytic sequence</scene> is PD<sup>91</sup> …E<sup>111</sup>AK. This motif is also responsible for Mg2+ binding(Asp90 and Glu111). [[Image:Catalytic 1ERI.png | thumb | The catalytic core of Eco RI with the target DNA]]<ref><br />
Structure and function of type II restriction endonucleases<br />
Alfred Pingoud, Albert Jeltsch<br />
Nucleic Acids Res. 2001 September 15; 29(18): 3705–3727.<br />
PMCID: PMC55916</ref><br />
<br />
<br />
== Application of EcoRI in molecular biology ==<br />
<br />
<br />
Type II restriction endonuclease like EcoRI are often used in molecular biology for their capacity to cut precisely DNA on specific restriction site. That makes them useful tools for gene cloning. By using two different restriction enzymes, it is possible to do directional cloning which is very important if you want to insert a gene in an expression vector.<br />
<br />
== Links ==<br />
[http://www.rcsb.org/pdb/explore/explore.do?structureId=1ERI] 1ERI in the Protein Database (PDB)<br />
<br />
== References ==<br />
<references/></div>Raphael Bilgerhttps://proteopedia.org/index.php?title=Sandbox_666&diff=1334071Sandbox 6662011-12-27T16:17:51Z<p>Raphael Bilger: </p>
<hr />
<div>Hello this Sandbox is reserved for a student project. It describes the structure of the endonuclese restriction enzyme Eco RI.<br />
{{STRUCTURE_1eri| PDB=1eri | SCENE= | size='500'}} <br />
== Presentation ==<br />
<br />
<br />
EcoRI is a type II restriction endonuclease. It recognizes and cleaves DNA on a specific palindromic sequence: GAATTC. EcoRI has been extracted from strain R Escherichia coli, a common bacterium which populates the intestine of mammalians. In bacteria, restriction enzymes protect the cell by cutting foreign DNA from bacteriophages (specific bacterial viruses).<br />
<br />
== Reaction ==<br />
<br />
<br />
EcoRI (E.C. 3.1.21.4) is a hydrolase and its substrate is a double-strand DNA molecule and two water molecules. For its catalytic activity, EcoRI needs a cofactor which is the divalent ion Mg<sup>2+</sup>. EcoRI hydrolyses the phosphodiester bond between the guanylic and adenylic residues resulting in 5’-phosphate sticky ends which are complementary.<br />
<br />
== Structure ==<br />
<br />
<br />
EcoRI is a homodimer, so it has two identical subunits (Representation of one <scene name='Sandbox_666/Monomer_structure/4'>subunit</scene>) of 31 kDa, but it possible to have homotetramers at high concentrations. The constitutive monomers are 276 amino acids long. EcoRI and all the other restriction enzymes show a common structural core which is a α/β domain. The constitutive subunits of EcoRI are organized into a single α/β domain (five stranded <scene name='Sandbox_666/B_sheet/1'>β</scene> sheet which is surrounded by <scene name='Sandbox_666/Helix/1'>α helices</scene> ). Four of these five β strands are parallel whereas the fourth (β4) is in an anti-parallel orientation to the others.<ref>Refinement of Eco RI endonuclease crystal structure: a revised protein chain tracing.<br />
Kim YC, Grable JC, Love R, Greene PJ, Rosenberg JM.</ref><br />
<br />
<br />
In the old model the N-terminal section of each subunit forms the inner arm which wraps around the DNA molecule (The arm brings the DNA molecule to the catalytic cleft.).The new chain tracing, based on new elements of electron density and anew interpretation that alters the assignment of specific amino acid residues to some of the original features<ref>Refinement of Eco RI endonuclease crystal structure: a revised protein chain tracing.<br />
Kim YC, Grable JC, Love R, Greene PJ, Rosenberg JM.</ref>. In the new model,the Inner arm is the extended chain motif (Met<sup>137</sup>to Ala<sup>142</sup>)is a segment of extended polypeptide chain that runs through the major groove of the DNA, roughly parallel to the DNA backbone<ref>Refinement of Eco RI endonuclease crystal structure: a revised protein chain tracing.<br />
Kim YC, Grable JC, Love R, Greene PJ, Rosenberg JM.</ref>. The Outer arm is composed of two minor β strands linked together by a loop (the outer arm is 14 amino acids long, four of these amino acids belong to the loop).<br />
<br />
<br />
The specific recognition of EcoRI of the GAATTC sequence is mediated by twelve hydrogen bonds (six bonds per subunit) originating from α helical recognition modules. Three amino acids are responsible for the recognition:Arg<sup>200</sup>, Glu<sup>144</sup> and Arg<sup>145</sup>. These aminoacids are shown in red <scene name='Sandbox_666/Grey_protein/3'>here</scene> .Each residue form two hydrogen bonds with Guanine and the adjacent Adenosine residues respectively. <br />
<br />
<br />
The reaction is due to a catalytic sequence motif which is found in most type II restriction endonucleases: the PD…D/EXK motif. For EcoRI, this catalytic sequence is PD<sup>91</sup> …E<sup>111</sup>AK.<scene name='Sandbox_666/Catalytic_core/3'>The catalytic core is shown here in red</scene>. This motif is also responsible for Mg2+ binding(Asp90 and Glu111). [[Image:Catalytic 1ERI.png | thumb | The catalytic core of Eco RI with the target DNA]]<ref><br />
Structure and function of type II restriction endonucleases<br />
Alfred Pingoud, Albert Jeltsch<br />
Nucleic Acids Res. 2001 September 15; 29(18): 3705–3727.<br />
PMCID: PMC55916</ref><br />
<br />
<br />
== Application of EcoRI in molecular biology ==<br />
<br />
<br />
Type II restriction endonuclease like EcoRI are often used in molecular biology for their capacity to cut precisely DNA on specific restriction site. That makes them useful tools for gene cloning. By using two different restriction enzymes, it is possible to do directional cloning which is very important if you want to insert a gene in an expression vector.<br />
<br />
== Links ==<br />
[http://www.rcsb.org/pdb/explore/explore.do?structureId=1ERI] 1ERI in the Protein Database (PDB)<br />
<br />
== References ==<br />
<references/></div>Raphael Bilgerhttps://proteopedia.org/index.php?title=Sandbox_666&diff=1333172Sandbox 6662011-12-19T12:51:30Z<p>Raphael Bilger: </p>
<hr />
<div>Hello this Sandbox is reserved for a student project<br />
{{STRUCTURE_1eri| PDB=1eri | SCENE= | size='500'}} <br />
== Presentation ==<br />
<br />
<br />
EcoRI is a type II restriction endonuclease. It recognizes and cleaves DNA on a specific palindromic sequence: GAATTC. EcoRI has been extracted from strain R Escherichia coli, a common bacterium which populates the intestine of mammalians. In bacteria, restriction enzymes protect the cell by cutting foreign DNA from bacteriophages (specific bacterial viruses).<br />
<br />
== Reaction ==<br />
<br />
<br />
EcoRI (E.C. 3.1.21.4) is a hydrolase and its substrate is a double-strand DNA molecule and two water molecules. For its catalytic activity, EcoRI needs a cofactor which is the divalent ion Mg<sup>2+</sup>. EcoRI hydrolyses the phosphodiester bond between the guanylic and adenylic residues resulting in 5’-phosphate sticky ends which are complementary.<br />
<br />
== Structure ==<br />
<br />
<br />
EcoRI is a homodimer, so it has two identical subunits (Representation of one <scene name='Sandbox_666/Monomer_structure/4'>subunit</scene>) of 31 kDa, but it possible to have homotetramers at high concentrations. The constitutive monomers are 276 amino acids long. EcoRI and all the other restriction enzymes show a common structural core which is a α/β domain. The constitutive subunits of EcoRI are organized into a single α/β domain (five stranded <scene name='Sandbox_666/B_sheet/1'>β</scene> sheet which is surrounded by <scene name='Sandbox_666/Helix/1'>α helices</scene> ). Four of these five β strands are parallel whereas the fourth (β4) is in an anti-parallel orientation to the others.<ref>Refinement of Eco RI endonuclease crystal structure: a revised protein chain tracing.<br />
Kim YC, Grable JC, Love R, Greene PJ, Rosenberg JM.</ref><br />
<br />
<br />
In the old model the N-terminal section of each subunit forms the inner arm which wraps around the DNA molecule (The arm brings the DNA molecule to the catalytic cleft.).The new chain tracing, based on new elements of electron density and anew interpretation that alters the assignment of specific amino acid residues to some of the original features<ref>Refinement of Eco RI endonuclease crystal structure: a revised protein chain tracing.<br />
Kim YC, Grable JC, Love R, Greene PJ, Rosenberg JM.</ref>. In the new model,the Inner arm is the extended chain motif (Met<sup>137</sup>to Ala<sup>142</sup>)is a segment of extended polypeptide chain that runs through the major groove of the DNA, roughly parallel to the DNA backbone<ref>Refinement of Eco RI endonuclease crystal structure: a revised protein chain tracing.<br />
Kim YC, Grable JC, Love R, Greene PJ, Rosenberg JM.</ref>. The Outer arm is composed of two minor β strands linked together by a loop (the outer arm is 14 amino acids long, four of these amino acids belong to the loop).<br />
<br />
<br />
The specific recognition of EcoRI of the GAATTC sequence is mediated by twelve hydrogen bonds (six bonds per subunit) originating from α helical recognition modules. Three amino acids are responsible for the recognition:Arg<sup>200</sup>, Glu<sup>144</sup> and Arg<sup>145</sup>. These aminoacids are shown in red <scene name='Sandbox_666/Grey_protein/3'>here</scene> .Each residue form two hydrogen bonds with Guanine and the adjacent Adenosine residues respectively. <br />
<br />
<br />
The reaction is due to a catalytic sequence motif which is found in most type II restriction endonucleases: the PD…D/EXK motif. For EcoRI, this catalytic sequence is PD<sup>91</sup> …E<sup>111</sup>AK.<scene name='Sandbox_666/Catalytic_core/3'>The catalytic core is shown here in red</scene>. This motif is also responsible for Mg2+ binding(Asp90 and Glu111). [[Image:Catalytic 1ERI.png | thumb | The catalytic core of Eco RI with the target DNA]]<ref><br />
Structure and function of type II restriction endonucleases<br />
Alfred Pingoud, Albert Jeltsch<br />
Nucleic Acids Res. 2001 September 15; 29(18): 3705–3727.<br />
PMCID: PMC55916</ref><br />
<br />
<br />
== Application of EcoRI in molecular biology ==<br />
<br />
<br />
Type II restriction endonuclease like EcoRI are often used in molecular biology for their capacity to cut precisely DNA on specific restriction site. That makes them useful tools for gene cloning. By using two different restriction enzymes, it is possible to do directional cloning which is very important if you want to insert a gene in an expression vector.<br />
<br />
== Links ==<br />
[http://www.rcsb.org/pdb/explore/explore.do?structureId=1ERI] 1ERI in the Protein Database (PDB)<br />
<br />
== References ==<br />
<references/></div>Raphael Bilgerhttps://proteopedia.org/index.php?title=Sandbox_666&diff=1333171Sandbox 6662011-12-19T12:48:30Z<p>Raphael Bilger: </p>
<hr />
<div>Hello this Sandbox is reserved for a student project<br />
{{STRUCTURE_1eri| PDB=1eri | SCENE= | size='500'}} <br />
== Presentation ==<br />
<br />
<br />
EcoRI is a type II restriction endonuclease. It recognizes and cleaves DNA on a specific palindromic sequence: GAATTC. EcoRI has been extracted from strain R Escherichia coli, a common bacterium which populates the intestine of mammalians. In bacteria, restriction enzymes protect the cell by cutting foreign DNA from bacteriophages (specific bacterial viruses).<br />
<br />
== Reaction ==<br />
<br />
<br />
EcoRI (E.C. 3.1.21.4) is a hydrolase and its substrate is a double-strand DNA molecule and two water molecules. For its catalytic activity, EcoRI needs a cofactor which is the divalent ion Mg<sup>2+</sup>. EcoRI hydrolyses the phosphodiester bond between the guanylic and adenylic residues resulting in 5’-phosphate sticky ends which are complementary.<br />
<br />
== Structure ==<br />
<br />
In the CDD (Conserved Domain Database, almost the entire sequence of EcoRI contains a conserved domain which is the EcoRI Superfamily.<br />
<br />
EcoRI is a homodimer, so it has two identical subunits (Representation of one <scene name='Sandbox_666/Monomer_structure/4'>subunit</scene>) of 31 kDa, but it possible to have homotetramers at high concentrations. The constitutive monomers are 276 amino acids long. EcoRI and all the other restriction enzymes show a common structural core which is a α/β domain. The constitutive subunits of EcoRI are organized into a single α/β domain (five stranded <scene name='Sandbox_666/B_sheet/1'>β</scene> sheet which is surrounded by <scene name='Sandbox_666/Helix/1'>α helices</scene> ). Four of these five β strands are parallel whereas the fourth (β4) is in an anti-parallel orientation to the others.<ref>Refinement of Eco RI endonuclease crystal structure: a revised protein chain tracing.<br />
Kim YC, Grable JC, Love R, Greene PJ, Rosenberg JM.</ref><br />
<br />
<br />
In the old model the N-terminal section of each subunit forms the inner arm which wraps around the DNA molecule (The arm brings the DNA molecule to the catalytic cleft.).The new chain tracing, based on new elements of electron density and anew interpretation that alters the assignment of specific amino acid residues to some of the original features<ref>Refinement of Eco RI endonuclease crystal structure: a revised protein chain tracing.<br />
Kim YC, Grable JC, Love R, Greene PJ, Rosenberg JM.</ref>. In the new model,the Inner arm is the extended chain motif (Met<sup>137</sup>to Ala<sup>142</sup>)is a segment of extended polypeptide chain that runs through the major groove of the DNA, roughly parallel to the DNA backbone<ref>Refinement of Eco RI endonuclease crystal structure: a revised protein chain tracing.<br />
Kim YC, Grable JC, Love R, Greene PJ, Rosenberg JM.</ref>. The Outer arm is composed of two minor β strands linked together by a loop (the outer arm is 14 amino acids long, four of these amino acids belong to the loop).<br />
<br />
<br />
The specific recognition of EcoRI of the GAATTC sequence is mediated by twelve hydrogen bonds (six bonds per subunit) originating from α helical recognition modules. Three amino acids are responsible for the recognition:Arg<sup>200</sup>, Glu<sup>144</sup> and Arg<sup>145</sup>. These aminoacids are shown in red <scene name='Sandbox_666/Grey_protein/3'>here</scene> .Each residue form two hydrogen bonds with Guanine and the adjacent Adenosine residues respectively. <br />
<br />
<br />
The reaction is due to a catalytic sequence motif which is found in most type II restriction endonucleases: the PD…D/EXK motif. For EcoRI, this catalytic sequence is PD<sup>91</sup> …E<sup>111</sup>AK.<scene name='Sandbox_666/Catalytic_core/3'>The catalytic core is shown here in red</scene>. This motif is also responsible for Mg2+ binding(Asp90 and Glu111). [[Image:Catalytic 1ERI.png | thumb | The catalytic core of Eco RI with the target DNA]]<ref><br />
Structure and function of type II restriction endonucleases<br />
Alfred Pingoud, Albert Jeltsch<br />
Nucleic Acids Res. 2001 September 15; 29(18): 3705–3727.<br />
PMCID: PMC55916</ref><br />
<br />
<br />
== Application of EcoRI in molecular biology ==<br />
<br />
<br />
Type II restriction endonuclease like EcoRI are often used in molecular biology for their capacity to cut precisely DNA on specific restriction site. That makes them useful tools for gene cloning. By using two different restriction enzymes, it is possible to do directional cloning which is very important if you want to insert a gene in an expression vector.<br />
<br />
== Links ==<br />
[http://www.rcsb.org/pdb/explore/explore.do?structureId=1ERI] 1ERI in the Protein Database (PDB)<br />
<br />
== References ==<br />
<references/></div>Raphael Bilgerhttps://proteopedia.org/index.php?title=Sandbox_666&diff=1332945Sandbox 6662011-12-16T17:57:13Z<p>Raphael Bilger: </p>
<hr />
<div>Hello this Sandbox is reserved for a student project<br />
{{STRUCTURE_1eri| PDB=1eri | SCENE= | size='500'}} <br />
== Presentation ==<br />
<br />
<br />
EcoRI is a type II restriction endonuclease. It recognizes and cleaves DNA on a specific palindromic sequence: GAATTC. EcoRI has been extracted from strain R Escherichia coli, a common bacterium which populates the intestine of mammalians. In bacteria, restriction enzymes protect the cell by cutting foreign DNA from bacteriophages (specific bacterial viruses).<br />
<br />
== Reaction ==<br />
<br />
<br />
EcoRI (E.C. 3.1.21.4) is a hydrolase and its substrate is a double-strand DNA molecule and two water molecules. For its catalytic activity, EcoRI needs a cofactor which is the divalent ion Mg<sup>2+</sup>. EcoRI hydrolyses the phosphodiester bond between the guanylic and adenylic residues resulting in 5’-phosphate sticky ends which are complementary.<br />
<br />
== Structure ==<br />
<br />
<br />
EcoRI is a homodimer, so it has two identical subunits (Representation of one <scene name='Sandbox_666/Monomer_structure/4'>subunit</scene>) of 31 kDa, but it possible to have homotetramers at high concentrations. The constitutive monomers are 276 amino acids long. EcoRI and all the other restriction enzymes show a common structural core which is a α/β domain. The constitutive subunits of EcoRI are organized into a single α/β domain (five stranded <scene name='Sandbox_666/B_sheet/1'>β</scene> sheet which is surrounded by <scene name='Sandbox_666/Helix/1'>α helices</scene> ). Four of these five β strands are parallel whereas the fourth (β4) is in an anti-parallel orientation to the others.<ref>Refinement of Eco RI endonuclease crystal structure: a revised protein chain tracing.<br />
Kim YC, Grable JC, Love R, Greene PJ, Rosenberg JM.</ref><br />
<br />
<br />
'''The N-terminal section of each subunit forms, with a β-hairpin, an “arm” which wraps around the DNA molecule. (The arm brings the DNA molecule to the catalytic cleft.)'''<br />
<br />
<br />
The specific recognition of EcoRI of the GAATTC sequence is mediated by twelve hydrogen bonds (six bonds per subunit) originating from α helical recognition modules. Three amino acids are responsible for the recognition:Arg<sup>200</sup>, Glu<sup>144</sup> and Arg<sup>145</sup>. These aminoacids are shown in red <scene name='Sandbox_666/Grey_protein/3'>here</scene> .Each residue form two hydrogen bonds with Guanine and the adjacent Adenosine residues respectively. <br />
<br />
<br />
The reaction is due to a catalytic sequence motif which is found in most type II restriction endonucleases: the PD…D/EXK motif. For EcoRI, this catalytic sequence is PD<sup>91</sup> …E<sup>111</sup>AK.<scene name='Sandbox_666/Catalytic_core/3'>The catalytic core is shown here in red</scene>. This motif is also responsible for Mg2+ binding(Asp90 and Glu111). [[Image:Catalytic 1ERI.png | thumb | The catalytic core of Eco RI with the target DNA]]<ref><br />
Structure and function of type II restriction endonucleases<br />
Alfred Pingoud, Albert Jeltsch<br />
Nucleic Acids Res. 2001 September 15; 29(18): 3705–3727.<br />
PMCID: PMC55916</ref><br />
<br />
<br />
== Application of EcoRI in molecular biology ==<br />
<br />
<br />
Type II restriction endonuclease like EcoRI are often used in molecular biology for their capacity to cut precisely DNA on specific restriction site. That makes them useful tools for gene cloning. By using two different restriction enzymes, it is possible to do directional cloning which is very important if you want to insert a gene in an expression vector.<br />
<br />
== Links ==<br />
[http://www.rcsb.org/pdb/explore/explore.do?structureId=1ERI] 1ERI in the Protein Database (PDB)<br />
<br />
== References ==<br />
<references/></div>Raphael Bilgerhttps://proteopedia.org/index.php?title=Sandbox_666&diff=1332891Sandbox 6662011-12-16T14:20:30Z<p>Raphael Bilger: </p>
<hr />
<div>Hello this Sandbox is reserved for a student project<br />
{{STRUCTURE_1eri| PDB=1eri | SCENE= | size='500'}} <br />
== Presentation ==<br />
<br />
<br />
EcoRI is a type II restriction endonuclease. It recognizes and cleaves DNA on a specific palindromic sequence: GAATTC. EcoRI has been extracted from strain R Escherichia coli, a common bacterium which populates the intestine of mammalians. In bacteria, restriction enzymes protect the cell by cutting foreign DNA from bacteriophages (specific bacterial viruses).<br />
<br />
== Reaction ==<br />
<br />
<br />
EcoRI (E.C. 3.1.21.4) is a hydrolase and its substrate is a double-strand DNA molecule and two water molecules. For its catalytic activity, EcoRI needs a cofactor which is the divalent ion Mg<sup>2+</sup>. EcoRI hydrolyses the phosphodiester bond between the guanylic and adenylic residues resulting in 5’-phosphate sticky ends which are complementary.<br />
<br />
== Structure ==<br />
<br />
<br />
EcoRI is a homodimer, so it has two identical subunits (Representation of one <scene name='Sandbox_666/Monomer_structure/3'>subunit</scene>) of 31 kDa, but it possible to have homotetramers at high concentrations. The constitutive monomers are 276 amino acids long. EcoRI and all the other restriction enzymes show a common structural core which is a α/β domain. The constitutive subunits of EcoRI are organized into a single α/β domain (five stranded <scene name='Sandbox_666/B_sheet/1'>β</scene> sheet which is surrounded by <scene name='Sandbox_666/Helix/1'>α helices</scene> ). Four of these five β strands are parallel whereas the fourth (β4) is in an anti-parallel orientation to the others.<ref>Refinement of Eco RI endonuclease crystal structure: a revised protein chain tracing.<br />
Kim YC, Grable JC, Love R, Greene PJ, Rosenberg JM.</ref><br />
<br />
<br />
'''The N-terminal section of each subunit forms, with a β-hairpin, an “arm” which wraps around the DNA molecule. (The arm brings the DNA molecule to the catalytic cleft.)'''<br />
<br />
<br />
The specific recognition of EcoRI of the GAATTC sequence is mediated by twelve hydrogen bonds (six bonds per subunit) originating from α helical recognition modules. Three amino acids are responsible for the recognition:Arg<sup>200</sup>, Glu<sup>144</sup> and Arg<sup>145</sup>. These aminoacids are shown in red <scene name='Sandbox_666/Grey_protein/3'>here</scene> .Each residue form two hydrogen bonds with Guanine and the adjacent Adenosine residues respectively. <br />
<br />
<br />
The reaction is due to a catalytic sequence motif which is found in most type II restriction endonucleases: the PD…D/EXK motif. For EcoRI, this catalytic sequence is PD<sup>91</sup> …E<sup>111</sup>AK.<scene name='Sandbox_666/Catalytic_core/3'>The catalytic core is shown here in red</scene>. This motif is also responsible for Mg2+ binding(Asp90 and Glu111). [[Image:Catalytic 1ERI.png | thumb | The catalytic core of Eco RI with the target DNA]]<ref><br />
Structure and function of type II restriction endonucleases<br />
Alfred Pingoud, Albert Jeltsch<br />
Nucleic Acids Res. 2001 September 15; 29(18): 3705–3727.<br />
PMCID: PMC55916</ref><br />
<br />
<br />
== Application of EcoRI in molecular biology ==<br />
<br />
<br />
Type II restriction endonuclease like EcoRI are often used in molecular biology for their capacity to cut precisely DNA on specific restriction site. That makes them useful tools for gene cloning. By using two different restriction enzymes, it is possible to do directional cloning which is very important if you want to insert a gene in an expression vector.<br />
<br />
== Links ==<br />
[http://www.rcsb.org/pdb/explore/explore.do?structureId=1ERI] 1ERI in the Protein Database (PDB)<br />
<br />
== References ==<br />
<references/></div>Raphael Bilgerhttps://proteopedia.org/index.php?title=Sandbox_666&diff=1332890Sandbox 6662011-12-16T14:16:06Z<p>Raphael Bilger: </p>
<hr />
<div>Hello this Sandbox is reserved for a student project<br />
{{STRUCTURE_1eri| PDB=1eri | SCENE= | size='500'}} <br />
== Presentation ==<br />
<br />
<br />
EcoRI is a type II restriction endonuclease. It recognizes and cleaves DNA on a specific palindromic sequence: GAATTC. EcoRI has been extracted from strain R Escherichia coli, a common bacterium which populates the intestine of mammalians. In bacteria, restriction enzymes protect the cell by cutting foreign DNA from bacteriophages (specific bacterial viruses).<br />
<br />
== Reaction ==<br />
<br />
<br />
EcoRI (E.C. 3.1.21.4) is a hydrolase and its substrate is a double-strand DNA molecule and two water molecules. For its catalytic activity, EcoRI needs a cofactor which is the divalent ion Mg<sup>2+</sup>. EcoRI hydrolyses the phosphodiester bond between the guanylic and adenylic residues resulting in 5’-phosphate sticky ends which are complementary.<br />
<br />
== Structure ==<br />
<br />
<br />
EcoRI is a homodimer, so it has two identical subunits (Representation of one <scene name='Sandbox_666/Monomer_structure/3'>subunit</scene>) of 31 kDa, but it possible to have homotetramers at high concentrations. The constitutive monomers are 276 amino acids long. EcoRI and all the other restriction enzymes show a common structural core which is a α/β domain. The constitutive subunits of EcoRI are organized into a single α/β domain (five stranded <scene name='Sandbox_666/B_sheet/1'>β</scene> sheet which is surrounded by <scene name='Sandbox_666/Helix/1'>α helices</scene> ). Four of these five β strands are parallel whereas the fourth (β4) is in an anti-parallel orientation to the others.<ref>Refinement of Eco RI endonuclease crystal structure: a revised protein chain tracing.<br />
Kim YC, Grable JC, Love R, Greene PJ, Rosenberg JM.</ref><br />
<br />
<br />
The N-terminal section of each subunit forms, with a β-hairpin, an “arm” which wraps around the DNA molecule. (The arm brings the DNA molecule to the catalytic cleft.)<br />
<br />
<br />
The specific recognition of EcoRI of the GAATTC sequence is mediated by twelve hydrogen bonds (six bonds per subunit) originating from α helical recognition modules. Three amino acids are responsible for the recognition:Arg<sup>200</sup>, Glu<sup>144</sup> and Arg<sup>145</sup>. These aminoacids are shown in red <scene name='Sandbox_666/Grey_protein/3'>here</scene> .Each residue form two hydrogen bonds with Guanine and the adjacent Adenosine residues respectively. <br />
<br />
<br />
The reaction is due to a catalytic sequence motif which is found in most type II restriction endonucleases: the PD…D/EXK motif. For EcoRI, this catalytic sequence is PD<sup>91</sup> …E<sup>111</sup>AK.<scene name='Sandbox_666/Catalytic_core/3'>The catalytic core is shown here in red</scene>. This motif is also responsible for Mg2+ binding(Asp90 and Glu111). [[Image:Catalytic 1ERI.png | thumb | The catalytic core of Eco RI with the target DNA]]<ref><br />
Structure and function of type II restriction endonucleases<br />
Alfred Pingoud, Albert Jeltsch<br />
Nucleic Acids Res. 2001 September 15; 29(18): 3705–3727.<br />
PMCID: PMC55916</ref><br />
<br />
<br />
== Application of EcoRI in molecular biology ==<br />
<br />
<br />
Type II restriction endonuclease like EcoRI are often used in molecular biology for their capacity to cut precisely DNA on specific restriction site. That makes them useful tools for gene cloning. By using two different restriction enzymes, it is possible to do directional cloning which is very important if you want to insert a gene in an expression vector.<br />
<br />
== Links ==<br />
[http://www.rcsb.org/pdb/explore/explore.do?structureId=1ERI] 1ERI in the Protein Database (PDB)<br />
<br />
== References ==<br />
<references/><br />
<br />
<br />
<!-- <br />
Please use the "3D" button above this box to insert a Jmol applet (molecule) on this page.<br />
Or use the four-green-boxes-button to insert scrollable text adjacent<br />
to a Jmol applet. Check out the other buttons as well! <br />
--></div>Raphael Bilgerhttps://proteopedia.org/index.php?title=Sandbox_666&diff=1332887Sandbox 6662011-12-16T14:09:58Z<p>Raphael Bilger: </p>
<hr />
<div>Hello this Sandbox is reserved for a student project<br />
{{STRUCTURE_1eri| PDB=1eri | SCENE= | size='500'}} <br />
== Presentation ==<br />
<br />
<br />
EcoRI is a type II restriction endonuclease. It recognizes and cleaves DNA on a specific palindromic sequence: GAATTC. EcoRI has been extracted from strain R Escherichia coli, a common bacterium which populates the intestine of mammalians. In bacteria, restriction enzymes protect the cell by cutting foreign DNA from bacteriophages (specific bacterial viruses).<br />
<br />
== Reaction ==<br />
<br />
<br />
EcoRI (E.C. 3.1.21.4) is a hydrolase and its substrate is a double-strand DNA molecule and two water molecules. For its catalytic activity, EcoRI needs a cofactor which is the divalent ion Mg<sup>2+</sup>. EcoRI hydrolyses the phosphodiester bond between the guanylic and adenylic residues resulting in 5’-phosphate sticky ends which are complementary.<br />
<br />
== Structure ==<br />
<br />
<br />
EcoRI is a homodimer, so it has two identical subunits (Representation of one <scene name='Sandbox_666/Monomer_structure/3'>subunit</scene>) of 31 kDa, but it possible to have homotetramers at high concentrations. The constitutive monomers are 276 amino acids long. EcoRI and all the other restriction enzymes show a common structural core which is a α/β domain. The constitutive subunits of EcoRI are organized into a single α/β domain (five stranded <scene name='Sandbox_666/B_sheet/1'>β</scene> sheet which is surrounded by <scene name='Sandbox_666/Helix/1'>α helices</scene> ). Four of these five β strands are parallel whereas the fourth (β4) is in an anti-parallel orientation to the others.<ref>Refinement of Eco RI endonuclease crystal structure: a revised protein chain tracing.<br />
Kim YC, Grable JC, Love R, Greene PJ, Rosenberg JM.</ref><br />
<br />
<br />
The N-terminal section of each subunit forms, with a β-hairpin, an “arm” which wraps around the DNA molecule. (The arm brings the DNA molecule to the catalytic cleft.)<br />
<br />
<br />
The specific recognition of EcoRI of the GAATTC sequence is mediated by twelve hydrogen bonds (six bonds per subunit) originating from α helical recognition modules. Three amino acids are responsible for the recognition:Arg<sup>200</sup>, Glu<sup>144</sup> and Arg<sup>145</sup>. These aminoacids are shown in red <scene name='Sandbox_666/Grey_protein/3'>here</scene> .Each residue form two hydrogen bonds with Guanine and the adjacent Adenosine residues respectively. <br />
<br />
<br />
The reaction is due to a catalytic sequence motif which is found in most type II restriction endonucleases: the PD…D/EXK motif. For EcoRI, this catalytic sequence is PD<sup>91</sup> …E<sup>111</sup>AK.<scene name='Sandbox_666/Catalytic_core/3'>The catalytic core is shown here in red</scene>. This motif is also responsible for Mg2+ binding(Asp90 and Glu111). [[Image:Catalytic 1ERI.png | thumb | The catalytic core of Eco RI with the target DNA]]<ref><br />
Structure and function of type II restriction endonucleases<br />
Alfred Pingoud, Albert Jeltsch<br />
Nucleic Acids Res. 2001 September 15; 29(18): 3705–3727.<br />
PMCID: PMC55916</ref><br />
<br />
<br />
== Application of EcoRI in molecular biology ==<br />
<br />
<br />
Type II restriction endonuclease like EcoRI are often used in molecular biology for their capacity to cut precisely DNA on specific restriction site. That makes them useful tools for gene cloning. By using two different restriction enzymes, it is possible to do directional cloning which is very important if you want to insert a gene in an expression vector.<br />
<br />
== Links ==<br />
[http://proteopedia.org/wiki/index.php?title=Sandbox_666&action=submit]<br />
== References ==<br />
<references/><br />
<br />
<br />
<!-- <br />
Please use the "3D" button above this box to insert a Jmol applet (molecule) on this page.<br />
Or use the four-green-boxes-button to insert scrollable text adjacent<br />
to a Jmol applet. Check out the other buttons as well! <br />
--></div>Raphael Bilgerhttps://proteopedia.org/index.php?title=File:Catalytic_1ERI.png&diff=1332873File:Catalytic 1ERI.png2011-12-16T13:11:38Z<p>Raphael Bilger: Catalytic core of 1ERI with DNA</p>
<hr />
<div>Catalytic core of 1ERI with DNA</div>Raphael Bilgerhttps://proteopedia.org/index.php?title=File:EcoRI_catalytic.mol&diff=1332868File:EcoRI catalytic.mol2011-12-16T13:03:00Z<p>Raphael Bilger: Picture of 1ERI</p>
<hr />
<div>Picture of 1ERI</div>Raphael Bilgerhttps://proteopedia.org/index.php?title=Sandbox_666&diff=1332817Sandbox 6662011-12-16T06:44:52Z<p>Raphael Bilger: </p>
<hr />
<div>Hello this Sandbox is reserved for a student project<br />
<Structure load='1ERI' size='500' frame='true' align='right' caption='3D structure of an Eco RI monomer' scene='Insert optional scene name here' /><br />
== Presentation ==<br />
<br />
<br />
EcoRI is a type II restriction endonuclease. It recognizes and cleaves DNA on a specific palindromic sequence: GAATTC. EcoRI has been extracted from strain R Escherichia coli, a common bacterium which populates the intestine of mammalians. In bacteria, restriction enzymes protect the cell by cutting foreign DNA from bacteriophages (specific bacterial viruses).<br />
<br />
== Reaction ==<br />
<br />
<br />
EcoRI (E.C. 3.1.21.4) is a hydrolase and its substrate is a double-strand DNA molecule and two water molecules. For its catalytic activity, EcoRI needs a cofactor which is the divalent ion Mg2+. EcoRI hydrolyses the phosphodiester bond between the guanylic and adenylic residues resulting in 5’-phosphate sticky ends which are complementary.<br />
<br />
== Structure ==<br />
<br />
<br />
EcoRI is a homodimer, so it has two identical subunits (Representation of one <scene name='Sandbox_666/Monomer_structure/3'>subunit</scene>) of 31 kDa, but it possible to have homotetramers at high concentrations. The constitutive monomers are 276 amino acids long. EcoRI and all the other restriction enzymes show a common structural core which is a α/β domain. The constitutive subunits of EcoRI are organized into a single α/β domain (five stranded <scene name='Sandbox_666/B_sheet/1'>β</scene> sheet which are surrounded on by <scene name='Sandbox_666/Helix/1'>α helices</scene> ). Four of these five β strands are parallel whereas the fourth (β4) is in an anti-parallel orientation to the others.<br />
<br />
<br />
The N-terminal section of each subunit forms, with a β-hairpin, an “arm” which wraps around the DNA molecule. (The arm brings the DNA molecule to the catalytic cleft.)<br />
<br />
<br />
The specific recognition of EcoRI of the GAATTC sequence is mediated by twelve hydrogen bonds (six bonds per subunit) originating from α helical recognition modules. Three amino acids are responsible for the recognition:Arg200, Glu144 and Arg145. These aminoacids are shown in red <scene name='Sandbox_666/Grey_protein/3'>here</scene> .Each residue form two hydrogen bonds with Guanine and the adjacent Adenosine residues respectively. <br />
<br />
<br />
The reaction is due to a catalytic sequence motif which is found in most type II restriction endonucleases: the PD…D/EXK motif. For EcoRI, this catalytic sequence is PD91 …E111AK.<scene name='Sandbox_666/Catalytic_core/3'>The catalytic core is shown here in red</scene>. This motif is also responsible for Mg2+ binding(Asp90 and Glu111).<br />
<br />
<br />
== Application of EcoRI in molecular biology ==<br />
<br />
<br />
Type II restriction endonuclease like EcoRI are often used in molecular biology for their capacity to cut precisely DNA on specific restriction site. That makes them useful tools for gene cloning. By using two different restriction enzymes, it is possible to do directional cloning which is very important if you want to insert a gene in an expression vector.<br />
<br />
<br />
== References ==<br />
<br />
<rRefinement of Eco RI endonuclease crystal structure: a revised protein chain tracing.<br />
Kim YC, Grable JC, Love R, Greene PJ, Rosenberg JM.<br />
Source<br />
Department of Biological Sciences, University of Pittsburgh, PA 15260./><br />
<br />
Structure and function of type II restriction endonucleases<br />
Alfred Pingoud, Albert Jeltsch<br />
Nucleic Acids Res. 2001 September 15; 29(18): 3705–3727.<br />
PMCID: PMC55916<br />
<br />
<!-- <br />
Please use the "3D" button above this box to insert a Jmol applet (molecule) on this page.<br />
Or use the four-green-boxes-button to insert scrollable text adjacent<br />
to a Jmol applet. Check out the other buttons as well! <br />
--></div>Raphael Bilgerhttps://proteopedia.org/index.php?title=Sandbox_666&diff=1332724Sandbox 6662011-12-15T19:15:18Z<p>Raphael Bilger: </p>
<hr />
<div>Hello this Sandbox is reserved for a student project<br />
<Structure load='1ERI' size='500' frame='true' align='right' caption='Insert caption here' scene='Insert optional scene name here' /><br />
== Presentation ==<br />
<br />
<br />
EcoRI is a type II restriction endonuclease. It recognizes and cleaves DNA on a specific palindromic sequence: GAATTC. EcoRI has been extracted from strain R Escherichia coli, a common bacterium which populates the intestine of mammalians. In bacteria, restriction enzymes protect the cell by cutting foreign DNA from bacteriophages (specific bacterial viruses).<br />
<br />
== Reaction ==<br />
<br />
<br />
EcoRI (E.C. 3.1.21.4) is a hydrolase and its substrate is a double-strand DNA molecule and two water molecules. For its catalytic activity, EcoRI needs a cofactor which is the divalent ion Mg2+. EcoRI hydrolyses the phosphodiester bond between the guanylic and adenylic residues resulting in 5’-phosphate sticky ends which are complementary.<br />
<br />
== Structure ==<br />
<br />
<br />
EcoRI is a homodimer, so it has two identical subunits (Representation of one <scene name='Sandbox_666/Monomer_structure/3'>subunit</scene>) of 31 kDa, but it possible to have homotetramers at high concentrations. The constitutive monomers are 276 amino acids long. EcoRI and all the other restriction enzymes show a common structural core which is a α/β domain. The constitutive subunits of EcoRI are organized into a single α/β domain (five stranded <scene name='Sandbox_666/B_sheet/1'>β</scene> sheet which are surrounded on by <scene name='Sandbox_666/Helix/1'>α helices</scene> ). Four of these five β strands are parallel whereas the fourth (β4) is in an anti-parallel orientation to the others.<br />
<br />
<br />
The N-terminal section of each subunit forms, with a β-hairpin, an “arm” which wraps around the DNA molecule. (The arm brings the DNA molecule to the catalytic cleft.)<br />
<br />
<br />
The specific recognition of EcoRI of the GAATTC sequence is mediated by twelve hydrogen bonds (six bonds per subunit) originating from α helical recognition modules. Three amino acids are responsible for the recognition:Arg200, Glu144 and Arg145. These aminoacids are shown in red <scene name='Sandbox_666/Grey_protein/3'>here</scene> .Each residue form two hydrogen bonds with Guanine and the adjacent Adenosine residues respectively. <br />
<br />
<br />
The reaction is due to a catalytic sequence motif which is found in most type II restriction endonucleases: the PD…D/EXK motif. For EcoRI, this catalytic sequence is PD91 …E111AK.<scene name='Sandbox_666/Catalytic_core/3'>The catalytic core is shown here in red</scene>. This motif is also responsible for Mg2+ binding(Asp90 and Glu111).<br />
<br />
<br />
== Application of EcoRI in molecular biology ==<br />
<br />
<br />
Type II restriction endonuclease like EcoRI are often used in molecular biology for their capacity to cut precisely DNA on specific restriction site. That makes them useful tools for gene cloning. By using two different restriction enzymes, it is possible to do directional cloning which is very important if you want to insert a gene in an expression vector.<br />
<br />
<br />
== References ==<br />
<br />
<rRefinement of Eco RI endonuclease crystal structure: a revised protein chain tracing.<br />
Kim YC, Grable JC, Love R, Greene PJ, Rosenberg JM.<br />
Source<br />
Department of Biological Sciences, University of Pittsburgh, PA 15260./><br />
<br />
Structure and function of type II restriction endonucleases<br />
Alfred Pingoud, Albert Jeltsch<br />
Nucleic Acids Res. 2001 September 15; 29(18): 3705–3727.<br />
PMCID: PMC55916<br />
<br />
<!-- <br />
Please use the "3D" button above this box to insert a Jmol applet (molecule) on this page.<br />
Or use the four-green-boxes-button to insert scrollable text adjacent<br />
to a Jmol applet. Check out the other buttons as well! <br />
--></div>Raphael Bilgerhttps://proteopedia.org/index.php?title=Sandbox_666&diff=1332723Sandbox 6662011-12-15T19:04:00Z<p>Raphael Bilger: </p>
<hr />
<div>Hello this Sandbox is reserved for a student project<br />
<Structure load='1ERI' size='500' frame='true' align='right' caption='Insert caption here' scene='Insert optional scene name here' /><br />
== Presentation ==<br />
<br />
<br />
EcoRI is a type II restriction endonuclease. It recognizes and cleaves DNA on a specific palindromic sequence: GAATTC. EcoRI has been extracted from strain R Escherichia coli, a common bacterium which populates the intestine of mammalians. In bacteria, restriction enzymes protect the cell by cutting foreign DNA from bacteriophages (specific bacterial viruses).<br />
<br />
== Reaction ==<br />
<br />
<br />
EcoRI (E.C. 3.1.21.4) is a hydrolase and its substrate is a double-strand DNA molecule and two water molecules. For its catalytic activity, EcoRI needs a cofactor which is the divalent ion Mg2+. EcoRI hydrolyses the phosphodiester bond between the guanylic and adenylic residues resulting in 5’-phosphate sticky ends which are complementary.<br />
<br />
== Structure ==<br />
<br />
<br />
EcoRI is a homodimer, so it has two identical subunits (Representation of one <scene name='Sandbox_666/Monomer_structure/3'>subunit</scene>, binding a single strand DNA molecule) of 31 kDa, but it possible to have homotetramers at high concentrations.. The constitutive monomers are 276 amino acids long. EcoRI and all the other restriction enzymes show a common structural core which is a α/β domain. The constitutive subunits of EcoRI are organized into a single α/β domain (five stranded <scene name='Sandbox_666/B_sheet/1'>β</scene> sheet which are surrounded on by <scene name='Sandbox_666/Helix/1'>α helices</scene> ). Four of these five β strands are parallel whereas the fourth (β4) is in an anti-parallel orientation to the others.<br />
<br />
<br />
The N-terminal section of each subunit forms, with a β-hairpin, an “arm” which wraps around the DNA molecule. (The arm brings the DNA molecule to the catalytic cleft.)<br />
<br />
<br />
The specific recognition of EcoRI of the GAATTC sequence is mediated by twelve hydrogen bonds (six bonds per subunit) originating from α helical recognition modules. Three amino acids are responsible for the recognition:Arg200, Glu144 and Arg145. These aminoacids are shown in red <scene name='Sandbox_666/Grey_protein/3'>here</scene> .Each residue form two hydrogen bonds with Guanine and the adjacent Adenosine residues respectively. <br />
<br />
<br />
The reaction is due to a catalytic sequence motif which is found in most type II restriction endonucleases: the PD…D/EXK motif. For EcoRI, this catalytic sequence is PD91 …E111AK.<scene name='Sandbox_666/Catalytic_core/3'>The catalytic core is shown here in red</scene>. This motif is also responsible for Mg2+ binding(Asp90 and Glu111).<br />
<br />
<br />
== Application of EcoRI in molecular biology ==<br />
<br />
<br />
Type II restriction endonuclease like EcoRI are often used in molecular biology for their capacity to cut precisely DNA on specific restriction site. That makes them useful tools for gene cloning. By using two different restriction enzymes, it is possible to do directional cloning which is very important if you want to insert a gene in an expression vector.<br />
<br />
<br />
== References ==<br />
<br />
<rRefinement of Eco RI endonuclease crystal structure: a revised protein chain tracing.<br />
Kim YC, Grable JC, Love R, Greene PJ, Rosenberg JM.<br />
Source<br />
Department of Biological Sciences, University of Pittsburgh, PA 15260./><br />
<br />
Structure and function of type II restriction endonucleases<br />
Alfred Pingoud, Albert Jeltsch<br />
Nucleic Acids Res. 2001 September 15; 29(18): 3705–3727.<br />
PMCID: PMC55916<br />
<br />
<!-- <br />
Please use the "3D" button above this box to insert a Jmol applet (molecule) on this page.<br />
Or use the four-green-boxes-button to insert scrollable text adjacent<br />
to a Jmol applet. Check out the other buttons as well! <br />
--></div>Raphael Bilgerhttps://proteopedia.org/index.php?title=Sandbox_666&diff=1332721Sandbox 6662011-12-15T19:01:04Z<p>Raphael Bilger: </p>
<hr />
<div>Hello this Sandbox is reserved for a student project<br />
<Structure load='1ERI' size='500' frame='true' align='right' caption='Insert caption here' scene='Insert optional scene name here' /><br />
== Presentation ==<br />
<br />
<br />
EcoRI is a type II restriction endonuclease. It recognizes and cleaves DNA on a specific palindromic sequence: GAATTC. EcoRI has been extracted from strain R Escherichia coli, a common bacterium which populates the intestine of mammalians. In bacteria, restriction enzymes protect the cell by cutting foreign DNA from bacteriophages (specific bacterial viruses).<br />
<br />
== Reaction ==<br />
<br />
<br />
EcoRI (E.C. 3.1.21.4) is a hydrolase and its substrate is a double-strand DNA molecule and two water molecules. For its catalytic activity, EcoRI needs a cofactor which is the divalent ion Mg2+. EcoRI hydrolyses the phosphodiester bond between the guanylic and adenylic residues resulting in 5’-phosphate sticky ends which are complementary.<br />
<br />
== Structure ==<br />
<br />
<br />
EcoRI is a homodimer, so it has two identical subunits (<scene name='Sandbox_666/Monomer_structure/3'>monomer strucure shown here</scene>) of 31 kDa, but it possible to have homotetramers at high concentrations.. The constitutive monomers are 276 amino acids long. EcoRI and all the other restriction enzymes show a common structural core which is a α/β domain. The constitutive subunits of EcoRI are organized into a single α/β domain (five stranded <scene name='Sandbox_666/B_sheet/1'>β</scene> sheet which are surrounded on by <scene name='Sandbox_666/Helix/1'>α helices</scene> ). Four of these five β strands are parallel whereas the fourth (β4) is in an anti-parallel orientation to the others.<br />
<br />
<br />
The N-terminal section of each subunit forms, with a β-hairpin, an “arm” which wraps around the DNA molecule. (The arm brings the DNA molecule to the catalytic cleft.)<br />
<br />
<br />
The specific recognition of EcoRI of the GAATTC sequence is mediated by twelve hydrogen bonds (six bonds per subunit) originating from α helical recognition modules. Three amino acids are responsible for the recognition:Arg200, Glu144 and Arg145. These aminoacids are shown in red <scene name='Sandbox_666/Grey_protein/3'>here</scene> .Each residue form two hydrogen bonds with Guanine and the adjacent Adenosine residues respectively. <br />
<br />
<br />
The reaction is due to a catalytic sequence motif which is found in most type II restriction endonucleases: the PD…D/EXK motif. For EcoRI, this catalytic sequence is PD91 …E111AK.<scene name='Sandbox_666/Catalytic_core/3'>The catalytic core is shown here in red</scene>. This motif is also responsible for Mg2+ binding(Asp90 and Glu111).<br />
<br />
<br />
== Application of EcoRI in molecular biology ==<br />
<br />
<br />
Type II restriction endonuclease like EcoRI are often used in molecular biology for their capacity to cut precisely DNA on specific restriction site. That makes them useful tools for gene cloning. By using two different restriction enzymes, it is possible to do directional cloning which is very important if you want to insert a gene in an expression vector.<br />
<br />
<br />
== References ==<br />
<br />
<rRefinement of Eco RI endonuclease crystal structure: a revised protein chain tracing.<br />
Kim YC, Grable JC, Love R, Greene PJ, Rosenberg JM.<br />
Source<br />
Department of Biological Sciences, University of Pittsburgh, PA 15260./><br />
<br />
Structure and function of type II restriction endonucleases<br />
Alfred Pingoud, Albert Jeltsch<br />
Nucleic Acids Res. 2001 September 15; 29(18): 3705–3727.<br />
PMCID: PMC55916<br />
<br />
<!-- <br />
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--></div>Raphael Bilgerhttps://proteopedia.org/index.php?title=Sandbox_666&diff=1332720Sandbox 6662011-12-15T18:59:14Z<p>Raphael Bilger: </p>
<hr />
<div>Hello this Sandbox is reserved for a student project<br />
<Structure load='1ERI' size='500' frame='true' align='right' caption='Insert caption here' scene='Insert optional scene name here' /><br />
== Presentation ==<br />
<br />
<br />
EcoRI is a type II restriction endonuclease. It recognizes and cleaves DNA on a specific palindromic sequence: GAATTC. EcoRI has been extracted from strain R Escherichia coli, a common bacterium which populates the intestine of mammalians. In bacteria, restriction enzymes protect the cell by cutting foreign DNA from bacteriophages (specific bacterial viruses).<br />
<br />
== Reaction ==<br />
<br />
<br />
EcoRI (E.C. 3.1.21.4) is a hydrolase and its substrate is a double-strand DNA molecule and two water molecules. For its catalytic activity, EcoRI needs a cofactor which is the divalent ion Mg2+. EcoRI hydrolyses the phosphodiester bond between the guanylic and adenylic residues resulting in 5’-phosphate sticky ends which are complementary.<br />
<br />
== Structure ==<br />
<br />
<br />
EcoRI is a homodimer, so it has two identical subunits (<scene name='Sandbox_666/Monomer_structure/3'>monomer strucure shown here</scene>) of 31 kDa, but it possible to have homotetramers at high concentrations.. The constitutive monomers are 276 amino acids long. EcoRI and all the other restriction enzymes show a common structural core which is a α/β domain. The constitutive subunits of EcoRI are organized into a single α/β domain (<scene name='Sandbox_666/B_sheet/1'>five stranded β</scene> sheet which are surrounded on by <scene name='Sandbox_666/Helix/1'>α helices</scene> ). Four of these five β strands are parallel whereas the fourth (β4) is in an anti-parallel orientation to the others.<br />
<br />
<br />
The N-terminal section of each subunit forms, with a β-hairpin, an “arm” which wraps around the DNA molecule. (The arm brings the DNA molecule to the catalytic cleft.)<br />
<br />
<br />
The specific recognition of EcoRI of the GAATTC sequence is mediated by twelve hydrogen bonds (six bonds per subunit) originating from α helical recognition modules. Three amino acids are responsible for the recognition:Arg200, Glu144 and Arg145. These aminoacids are shown in red <scene name='Sandbox_666/Grey_protein/3'>here</scene> .Each residue form two hydrogen bonds with Guanine and the adjacent Adenosine residues respectively. <br />
<br />
<br />
The reaction is due to a catalytic sequence motif which is found in most type II restriction endonucleases: the PD…D/EXK motif. For EcoRI, this catalytic sequence is PD91 …E111AK.<scene name='Sandbox_666/Catalytic_core/3'>The catalytic core is shown here in red</scene>. This motif is also responsible for Mg2+ binding(Asp90 and Glu111).<br />
<br />
<br />
== Application of EcoRI in molecular biology ==<br />
<br />
<br />
Type II restriction endonuclease like EcoRI are often used in molecular biology for their capacity to cut precisely DNA on specific restriction site. That makes them useful tools for gene cloning. By using two different restriction enzymes, it is possible to do directional cloning which is very important if you want to insert a gene in an expression vector.<br />
<br />
<br />
== References ==<br />
<br />
<rRefinement of Eco RI endonuclease crystal structure: a revised protein chain tracing.<br />
Kim YC, Grable JC, Love R, Greene PJ, Rosenberg JM.<br />
Source<br />
Department of Biological Sciences, University of Pittsburgh, PA 15260./><br />
<br />
Structure and function of type II restriction endonucleases<br />
Alfred Pingoud, Albert Jeltsch<br />
Nucleic Acids Res. 2001 September 15; 29(18): 3705–3727.<br />
PMCID: PMC55916<br />
<br />
<!-- <br />
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--></div>Raphael Bilgerhttps://proteopedia.org/index.php?title=Sandbox_20012&diff=1332698Sandbox 200122011-12-15T17:12:38Z<p>Raphael Bilger: </p>
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<div><!-- <br />
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<Structure load='3g04' size='500' frame='true' align='right' caption='Insert caption here' scene='Insert optional scene name here' /><br />
<scene name='Sandbox_20012/Protein/1'>chaine A</scene></div>Raphael Bilgerhttps://proteopedia.org/index.php?title=Sandbox_20012&diff=1332697Sandbox 200122011-12-15T17:07:48Z<p>Raphael Bilger: New page: <!-- Please use the "3D" button above this box to insert a Jmol applet (molecule) on this page. Or use the four-green-boxes-button to insert scrollable text adjacent to a Jmol applet. Che...</p>
<hr />
<div><!-- <br />
Please use the "3D" button above this box to insert a Jmol applet (molecule) on this page.<br />
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<br />
<Structure load='3g04' size='500' frame='true' align='right' caption='Insert caption here' scene='Insert optional scene name here' /></div>Raphael Bilgerhttps://proteopedia.org/index.php?title=Sandbox_666&diff=1332694Sandbox 6662011-12-15T16:48:10Z<p>Raphael Bilger: </p>
<hr />
<div>Hello this Sandbox is reserved for a student project<br />
<Structure load='1ERI' size='500' frame='true' align='right' caption='Insert caption here' scene='Insert optional scene name here' /><br />
== Presentation ==<br />
<br />
<br />
EcoRI is a type II restriction endonuclease. It recognizes and cleaves DNA on a specific palindromic sequence: GAATTC. EcoRI has been extracted from strain R Escherichia coli, a common bacterium which populates the intestine of mammalians. In bacteria, restriction enzymes protect the cell by cutting foreign DNA from bacteriophages (specific bacterial viruses).<br />
<br />
== Reaction ==<br />
<br />
<br />
EcoRI (E.C. 3.1.21.4) is a hydrolase and its substrate is a double-strand DNA molecule and two water molecules. For its catalytic activity, EcoRI needs a cofactor which is the divalent ion Mg2+. EcoRI hydrolyses the phosphodiester bond between the guanylic and adenylic residues resulting in 5’-phosphate sticky ends which are complementary.<br />
<br />
== Structure ==<br />
<br />
<br />
EcoRI is a homodimer, so it has two identical subunits (<scene name='Sandbox_666/Monomer_structure/3'>monomer strucure shown here</scene>) of 31 kDa, but it possible to have homotetramers at high concentrations.. The constitutive monomers are 276 amino acids long. EcoRI and all the other restriction enzymes show a common structural core which is a α/β domain. The constitutive subunits of EcoRI are organized into a single α/β domain (five stranded β sheet which are surrounded on by α helices). Four of these five β strands are parallel whereas the fourth (β4) is in an anti-parallel orientation to the others.<br />
<br />
<br />
The N-terminal section of each subunit forms, with a β-hairpin, an “arm” which wraps around the DNA molecule. (The arm brings the DNA molecule to the catalytic cleft.)<br />
<br />
<br />
The specific recognition of EcoRI of the GAATTC sequence is mediated by twelve hydrogen bonds (six bonds per subunit) originating from α helical recognition modules. Three amino acids are responsible for the recognition:Arg200, Glu144 and Arg145. These aminoacids are shown in red <scene name='Sandbox_666/Grey_protein/3'>here</scene> .Each residue form two hydrogen bonds with Guanine and the adjacent Adenosine residues respectively. <br />
<br />
<br />
The reaction is due to a catalytic sequence motif which is found in most type II restriction endonucleases: the PD…D/EXK motif. For EcoRI, this catalytic sequence is PD91 …E111AK.<scene name='Sandbox_666/Catalytic_core/3'>The catalytic core is shown here in red</scene>. This motif is also responsible for Mg2+ binding(Asp90 and Glu111).<br />
<br />
<br />
== Application of EcoRI in molecular biology ==<br />
<br />
<br />
Type II restriction endonuclease like EcoRI are often used in molecular biology for their capacity to cut precisely DNA on specific restriction site. That makes them useful tools for gene cloning. By using two different restriction enzymes, it is possible to do directional cloning which is very important if you want to insert a gene in an expression vector.<br />
<br />
<br />
== References ==<br />
<br />
<rRefinement of Eco RI endonuclease crystal structure: a revised protein chain tracing.<br />
Kim YC, Grable JC, Love R, Greene PJ, Rosenberg JM.<br />
Source<br />
Department of Biological Sciences, University of Pittsburgh, PA 15260./><br />
<br />
Structure and function of type II restriction endonucleases<br />
Alfred Pingoud, Albert Jeltsch<br />
Nucleic Acids Res. 2001 September 15; 29(18): 3705–3727.<br />
PMCID: PMC55916<br />
<br />
<!-- <br />
Please use the "3D" button above this box to insert a Jmol applet (molecule) on this page.<br />
Or use the four-green-boxes-button to insert scrollable text adjacent<br />
to a Jmol applet. Check out the other buttons as well! <br />
--></div>Raphael Bilgerhttps://proteopedia.org/index.php?title=Sandbox_666&diff=1332688Sandbox 6662011-12-15T16:32:44Z<p>Raphael Bilger: </p>
<hr />
<div>Hello this Sandbox is reserved for a student project<br />
<Structure load='1ERI' size='500' frame='true' align='right' caption='Insert caption here' scene='Insert optional scene name here' /><br />
== Presentation ==<br />
<br />
<br />
EcoRI is a type II restriction endonuclease. It recognizes and cleaves DNA on a specific palindromic sequence: GAATTC. EcoRI has been extracted from strain R Escherichia coli, a common bacterium which populates the intestine of mammalians. In bacteria, restriction enzymes protect the cell by cutting foreign DNA from bacteriophages (specific bacterial viruses).<br />
<br />
== Reaction ==<br />
<br />
<br />
EcoRI (E.C. 3.1.21.4) is a hydrolase and its substrate is a double-strand DNA molecule and two water molecules. For its catalytic activity, EcoRI needs a cofactor which is the divalent ion Mg2+. EcoRI hydrolyses the phosphodiester bond between the guanylic and adenylic residues resulting in 5’-phosphate sticky ends which are complementary.<br />
<br />
== Structure ==<br />
<br />
<br />
EcoRI is a homodimer, so it has two identical subunits (<scene name='Sandbox_666/Monomer_structure/3'>monomer strucure shown here</scene>) of 31 kDa, but it possible to have homotetramers at high concentrations.. The constitutive monomers are 276 amino acids long. EcoRI and all the other restriction enzymes show a common structural core which is a α/β domain. The constitutive subunits of EcoRI are organized into a single α/β domain (five stranded β sheet which are surrounded on by α helices). Four of these five β strands are parallel whereas the fourth (β4) is in an anti-parallel orientation to the others.<br />
<br />
<br />
<br />
The N-terminal section of each subunit forms, with a β-hairpin, an “arm” which wraps around the DNA molecule. (The arm brings the DNA molecule to the catalytic cleft.)<br />
<br />
<br />
<br />
The specific recognition of EcoRI of the GAATTC sequence is mediated by twelve hydrogen bonds (six bonds per subunit) originating from α helical recognition modules. Three amino acids are responsible for the recognition:Arg200, Glu144 and Arg145. These aminoacids are shown in red <scene name='Sandbox_666/Grey_protein/3'>here</scene> .Each residue form two hydrogen bonds with Guanine and the adjacent Adenosine residues respectively. <br />
<br />
<br />
<br />
The reaction is due to a catalytic sequence motif which is found in most type II restriction endonucleases: the PD…D/EXK motif. For EcoRI, this catalytic sequence is PD91 …E111AK.<scene name='Sandbox_666/Catalytic_core/3'>The catalytic core is shown here in red</scene>. This motif is also responsible for Mg2+ binding(Asp90 and Glu111).<br />
<br />
<br />
== Application of EcoRI in molecular biology ==<br />
<br />
<br />
Type II restriction endonuclease like EcoRI are often used in molecular biology for their capacity to cut precisely DNA on specific restriction site. That makes them useful tools for gene cloning. By using two different restriction enzymes, it is possible to do directional cloning which is very important if you want to insert a gene in an expression vector.<br />
<br />
<br />
== References ==<br />
<rRefinement of Eco RI endonuclease crystal structure: a revised protein chain tracing.<br />
Kim YC, Grable JC, Love R, Greene PJ, Rosenberg JM.<br />
Source<br />
Department of Biological Sciences, University of Pittsburgh, PA 15260./><br />
<Structure and function of type II restriction endonucleases<br />
Alfred Pingoud, Albert Jeltsch<br />
Nucleic Acids Res. 2001 September 15; 29(18): 3705–3727.<br />
PMCID: PMC55916/><br />
<!-- <br />
Please use the "3D" button above this box to insert a Jmol applet (molecule) on this page.<br />
Or use the four-green-boxes-button to insert scrollable text adjacent<br />
to a Jmol applet. Check out the other buttons as well! <br />
--></div>Raphael Bilgerhttps://proteopedia.org/index.php?title=Sandbox_666&diff=1332687Sandbox 6662011-12-15T16:18:02Z<p>Raphael Bilger: </p>
<hr />
<div>Hello this Sandbox is reserved for a student project<br />
<Structure load='1ERI' size='500' frame='true' align='right' caption='Insert caption here' scene='Insert optional scene name here' /><br />
== Presentation ==<br />
<br />
<br />
EcoRI is a type II restriction endonuclease. It recognizes and cleaves DNA on a specific palindromic sequence: GAATTC. EcoRI has been extracted from strain R Escherichia coli, a common bacterium which populates the intestine of mammalians. In bacteria, restriction enzymes protect the cell by cutting foreign DNA from bacteriophages (specific bacterial viruses).<br />
<br />
== Reaction ==<br />
<br />
<br />
EcoRI (E.C. 3.1.21.4) is a hydrolase and its substrate is a double-strand DNA molecule and two water molecules. For its catalytic activity, EcoRI needs a cofactor which is the divalent ion Mg2+. EcoRI hydrolyses the phosphodiester bond between the guanylic and adenylic residues resulting in 5’-phosphate sticky ends which are complementary.<br />
<br />
== Structure ==<br />
<br />
<br />
EcoRI is a homodimer, so it has two identical subunits (<scene name='Sandbox_666/Monomer_structure/3'>monomer strucure shown here</scene>) of 31 kDa, but it possible to have homotetramers at high concentrations.. The constitutive monomers are 276 amino acids long. EcoRI and all the other restriction enzymes show a common structural core which is a α/β domain. The constitutive subunits of EcoRI are organized into a single α/β domain (five stranded β sheet which are surrounded on by α helices). Four of these five β strands are parallel whereas the fourth (β4) is in an anti-parallel orientation to the others.<br />
<br />
<br />
<br />
The N-terminal section of each subunit forms, with a β-hairpin, an “arm” which wraps around the DNA molecule. (The arm brings the DNA molecule to the catalytic cleft.)<br />
<br />
<br />
<br />
The specific recognition of EcoRI of the GAATTC sequence is mediated by twelve hydrogen bonds (six bonds per subunit) originating from α helical recognition modules. Three amino acids are responsible for the recognition:Arg200, Glu144 and Arg145. These aminoacids are shown in red <scene name='Sandbox_666/Grey_protein/3'>here</scene> .Each residue form two hydrogen bonds with Guanine and the adjacent Adenosine residues respectively. <br />
<br />
<br />
<br />
The reaction is due to a catalytic sequence motif which is found in most type II restriction endonucleases: the PD…D/EXK motif. For EcoRI, this catalytic sequence is PD91 …E111AK.<scene name='Sandbox_666/Catalytic_core/3'>The catalytic core is shown here in red</scene>. This motif is also responsible for Mg2+ binding(Asp90 and Glu111).<br />
<br />
<br />
== Application of EcoRI in molecular biology ==<br />
<br />
<br />
Type II restriction endonuclease like EcoRI are often used in molecular biology for their capacity to cut precisely DNA on specific restriction site. That makes them useful tools for gene cloning. By using two different restriction enzymes, it is possible to do directional cloning which is very important if you want to insert a gene in an expression vector.<br />
<br />
<br />
== References ==<br />
<!-- <br />
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--></div>Raphael Bilgerhttps://proteopedia.org/index.php?title=Sandbox_666&diff=1332686Sandbox 6662011-12-15T16:13:48Z<p>Raphael Bilger: </p>
<hr />
<div>Hello this Sandbox is reserved for a student project<br />
<Structure load='1ERI' size='500' frame='true' align='right' caption='Insert caption here' scene='Insert optional scene name here' /><br />
== Presentation ==<br />
<br />
<br />
EcoRI is a type II restriction endonuclease. It recognizes and cleaves DNA on a specific palindromic sequence: GAATTC. EcoRI has been extracted from strain R Escherichia coli, a common bacterium which populates the intestine of mammalians. In bacteria, restriction enzymes protect the cell by cutting foreign DNA from bacteriophages (specific bacterial viruses).<br />
<br />
== Reaction ==<br />
<br />
EcoRI (E.C. 3.1.21.4) is a hydrolase and its substrate is a double-strand DNA molecule and two water molecules. For its catalytic activity, EcoRI needs a cofactor which is the divalent ion Mg2+. EcoRI hydrolyses the phosphodiester bond between the guanylic and adenylic residues resulting in 5’-phosphate sticky ends which are complementary.<br />
<br />
== Structure ==<br />
<br />
EcoRI is a homodimer, so it has two identical subunits (<scene name='Sandbox_666/Monomer_structure/3'>monomer strucure shown here</scene>) of 31 kDa, but it possible to have homotetramers at high concentrations.. The constitutive monomers are 276 amino acids long. EcoRI and all the other restriction enzymes show a common structural core which is a α/β domain. The constitutive subunits of EcoRI are organized into a single α/β domain (five stranded β sheet which are surrounded on by α helices). Four of these five β strands are parallel whereas the fourth (β4) is in an anti-parallel orientation to the others.<br />
<br />
The N-terminal section of each subunit forms, with a β-hairpin, an “arm” which wraps around the DNA molecule. (The arm brings the DNA molecule to the catalytic cleft.)<br />
<br />
The specific recognition of EcoRI of the GAATTC sequence is mediated by twelve hydrogen bonds (six bonds per subunit) originating from α helical recognition modules. Three amino acids are responsible for the recognition:Arg200, Glu144 and Arg145. These aminoacids are shown in red <scene name='Sandbox_666/Grey_protein/3'>here</scene> .Each residue form two hydrogen bonds with Guanine and the adjacent Adenosine residues respectively. <br />
<br />
The reaction is due to a catalytic sequence motif which is found in most type II restriction endonucleases: the PD…D/EXK motif. For EcoRI, this catalytic sequence is PD91 …E111AK.<scene name='Sandbox_666/Catalytic_core/3'>The catalytic core is shown here in red</scene>. This motif is also responsible for Mg2+ binding(Asp90 and Glu111).<br />
<br />
<br />
== Application of EcoRI in molecular biology ==<br />
<br />
Type II restriction endonuclease like EcoRI are often used in molecular biology for their capacity to cut precisely DNA on specific restriction site. That makes them useful tools for gene cloning. By using two different restriction enzymes, it is possible to do directional cloning which is very important if you want to insert a gene in an expression vector.<br />
<br />
<br />
== References ==<br />
<!-- <br />
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--></div>Raphael Bilgerhttps://proteopedia.org/index.php?title=Sandbox_666&diff=1332680Sandbox 6662011-12-15T15:42:22Z<p>Raphael Bilger: </p>
<hr />
<div>Hello this Sandbox is reserved for a student project<br />
<Structure load='1ERI' size='500' frame='true' align='right' caption='Insert caption here' scene='Insert optional scene name here' /><br />
== Presentation ==<br />
<br />
<br />
EcoRI is a type II restriction endonuclease. It recognizes and cleaves DNA on a specific palindromic sequence: GAATTC. EcoRI has been extracted from strain R Escherichia coli, a common bacterium which populates the intestine of mammalians. In bacteria, restriction enzymes protect the cell by cutting foreign DNA from bacteriophages (specific bacterial viruses).<br />
<br />
== Reaction ==<br />
<br />
EcoRI (E.C. 3.1.21.4) is a hydrolase and its substrate is a double-strand DNA molecule and two water molecules. For its catalytic activity, EcoRI needs a cofactor which is the divalent ion Mg2+. EcoRI hydrolyses the phosphodiester bond between the guanylic and adenylic residues resulting in 5’-phosphate sticky ends which are complementary.<br />
<br />
== Structure ==<br />
<br />
EcoRI is a homodimer, so it has two identical subunits (<scene name='Sandbox_666/Monomer_structure/3'>monomer strucure shown here</scene>) of 31 kDa, but it possible to have homotetramers at high concentrations.. The constitutive monomers are 276 amino acids long. EcoRI and all the other restriction enzymes show a common structural core which is a α/β domain. The constitutive subunits of EcoRI are organized into a single α/β domain (five stranded β sheet which are surrounded on by α helices). Four of these five β strands are parallel whereas the fourth (β4) is in an anti-parallel orientation to the others.<br />
<br />
The N-terminal section of each subunit forms, with a β-hairpin, an “arm” which wraps around the DNA molecule. (The arm brings the DNA molecule to the catalytic cleft.)<br />
<br />
The specific recognition of EcoRI of the GAATTC sequence is mediated by twelve hydrogen bonds (six bonds per subunit) originating from α helical recognition modules. Three amino acids are responsible for the recognition:Arg200, Glu144 and Arg145. These aminoacids are shown in red <scene name='Sandbox_666/Grey_protein/3'>here</scene> .Each residue form two hydrogen bonds with Guanine and the adjacent Adenosine residues respectively. <br />
<br />
The reaction is due to a catalytic sequence motif which is found in most type II restriction endonucleases: the PD…D/EXK motif. For EcoRI, this catalytic sequence is PD91 …E111AK.<scene name='Sandbox_666/Catalytic_core/3'>The catalytic core is shown here in red</scene>. This motif is also responsible for Mg2+ binding(Asp90 and Glu111).<br />
<br />
<br />
== Application of EcoRI in molecular biology ==<br />
<br />
Type II restriction endonuclease like EcoRI are often used in molecular biology for their capacity to cut precisely DNA on specific restriction site. That makes them useful tools for gene cloning. By using two different restriction enzymes, it is possible to do directional cloning which is very important if you want to insert a gene in an expression vector.<br />
<br />
<br />
== References ==<br />
<!-- <br />
Please use the "3D" button above this box to insert a Jmol applet (molecule) on this page.<br />
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--></div>Raphael Bilgerhttps://proteopedia.org/index.php?title=Sandbox_666&diff=1332678Sandbox 6662011-12-15T15:40:34Z<p>Raphael Bilger: </p>
<hr />
<div>Hello this Sandbox is reserved for a student project<br />
<Structure load='1ERI' size='500' frame='true' align='right' caption='Insert caption here' scene='Insert optional scene name here' /><br />
== Presentation ==<br />
<br />
<br />
EcoRI is a type II restriction endonuclease. It recognizes and cleaves DNA on a specific palindromic sequence: GAATTC. EcoRI has been extracted from strain R Escherichia coli, a common bacterium which populates the intestine of mammalians. In bacteria, restriction enzymes protect the cell by cutting foreign DNA from bacteriophages (specific bacterial viruses).<br />
<br />
== Reaction ==<br />
<br />
EcoRI (E.C. 3.1.21.4) is a hydrolase and its substrate is a double-strand DNA molecule and two water molecules. For its catalytic activity, EcoRI needs a cofactor which is the divalent ion Mg2+. EcoRI hydrolyses the phosphodiester bond between the guanylic and adenylic residues resulting in 5’-phosphate sticky ends which are complementary.<br />
<br />
== Structure ==<br />
<br />
EcoRI is a homodimer, so it has two identical subunits (<scene name='Sandbox_666/Monomer_structure/3'>monomer strucure shown here</scene>) of 31 kDa, but it possible to have homotetramers at high concentrations.. The constitutive monomers are 276 amino acids long. EcoRI and all the other restriction enzymes show a common structural core which is a α/β domain. The constitutive subunits of EcoRI are organized into a single α/β domain (five stranded β sheet which are surrounded on by α helices). Four of these five β strands are parallel whereas the fourth (β4) is in an anti-parallel orientation to the others.<br />
<br />
The N-terminal section of each subunit forms, with a β-hairpin, an “arm” which wraps around the DNA molecule. (The arm brings the DNA molecule to the catalytic cleft.)<br />
<br />
The specific recognition of EcoRI of the GAATTC sequence is mediated by twelve hydrogen bonds (six bonds per subunit) originating from α helical recognition modules. Three amino acids are responsible for the recognition:Arg200, Glu144 and Arg145<scene name='Sandbox_666/Grey_protein/3'>TextToBeDisplayed</scene> each residue form two hydrogen bonds with Guanine and the adjacent Adenosine residues respectively. <br />
<br />
The reaction is due to a catalytic sequence motif which is found in most type II restriction endonucleases: the PD…D/EXK motif. For EcoRI, this catalytic sequence is PD91 …E111AK.<scene name='Sandbox_666/Catalytic_core/3'>The catalytic core is shown here in red</scene>. This motif is also responsible for Mg2+ binding(Asp90 and Glu111).<br />
<br />
<br />
== Application of EcoRI in molecular biology ==<br />
<br />
Type II restriction endonuclease like EcoRI are often used in molecular biology for their capacity to cut precisely DNA on specific restriction site. That makes them useful tools for gene cloning. By using two different restriction enzymes, it is possible to do directional cloning which is very important if you want to insert a gene in an expression vector.<br />
<br />
<br />
== References ==<br />
<!-- <br />
Please use the "3D" button above this box to insert a Jmol applet (molecule) on this page.<br />
Or use the four-green-boxes-button to insert scrollable text adjacent<br />
to a Jmol applet. Check out the other buttons as well! <br />
--></div>Raphael Bilgerhttps://proteopedia.org/index.php?title=Sandbox_666&diff=1332676Sandbox 6662011-12-15T15:37:06Z<p>Raphael Bilger: </p>
<hr />
<div>Hello this Sandbox is reserved for a student project<br />
<Structure load='1ERI' size='500' frame='true' align='right' caption='Insert caption here' scene='Insert optional scene name here' /><br />
== Presentation ==<br />
<br />
<br />
EcoRI is a type II restriction endonuclease. It recognizes and cleaves DNA on a specific palindromic sequence: GAATTC. EcoRI has been extracted from strain R Escherichia coli, a common bacterium which populates the intestine of mammalians. In bacteria, restriction enzymes protect the cell by cutting foreign DNA from bacteriophages (specific bacterial viruses).<br />
<br />
== Reaction ==<br />
<br />
EcoRI (E.C. 3.1.21.4) is a hydrolase and its substrate is a double-strand DNA molecule and two water molecules. For its catalytic activity, EcoRI needs a cofactor which is the divalent ion Mg2+. EcoRI hydrolyses the phosphodiester bond between the guanylic and adenylic residues resulting in 5’-phosphate sticky ends which are complementary.<br />
<br />
== Structure ==<br />
<br />
EcoRI is a homodimer, so it has two identical subunits (<scene name='Sandbox_666/Monomer_structure/3'>monomer strucure shown here</scene>) of 31 kDa, but it possible to have homotetramers at high concentrations.. The constitutive monomers are 276 amino acids long. EcoRI and all the other restriction enzymes show a common structural core which is a α/β domain. The constitutive subunits of EcoRI are organized into a single α/β domain (five stranded β sheet which are surrounded on by α helices). Four of these five β strands are parallel whereas the fourth (β4) is in an anti-parallel orientation to the others.<br />
<br />
The N-terminal section of each subunit forms, with a β-hairpin, an “arm” which wraps around the DNA molecule. (The arm brings the DNA molecule to the catalytic cleft.)<br />
<br />
The specific recognition of EcoRI of the GAATTC sequence is mediated by twelve hydrogen bonds (six bonds per subunit) originating from α helical recognition modules. Three amino acids are responsible for the recognition:<scene name='Sandbox_666/Grey_protein/2'>Arg200, Glu144 and Arg145/scene>, each residue form two hydrogen bonds with Guanine and the adjacent Adenosine residues respectively. <br />
<br />
The reaction is due to a catalytic sequence motif which is found in most type II restriction endonucleases: the PD…D/EXK motif. For EcoRI, this catalytic sequence is PD91 …E111AK.<scene name='Sandbox_666/Catalytic_core/3'>The catalytic core is shown here in red</scene>. This motif is also responsible for Mg2+ binding(Asp90 and Glu111).<br />
<br />
<br />
== Application of EcoRI in molecular biology ==<br />
<br />
Type II restriction endonuclease like EcoRI are often used in molecular biology for their capacity to cut precisely DNA on specific restriction site. That makes them useful tools for gene cloning. By using two different restriction enzymes, it is possible to do directional cloning which is very important if you want to insert a gene in an expression vector.<br />
<br />
<br />
== References ==<br />
<!-- <br />
Please use the "3D" button above this box to insert a Jmol applet (molecule) on this page.<br />
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to a Jmol applet. Check out the other buttons as well! <br />
--></div>Raphael Bilgerhttps://proteopedia.org/index.php?title=Sandbox_666&diff=1332668Sandbox 6662011-12-15T15:23:51Z<p>Raphael Bilger: </p>
<hr />
<div>Hello this Sandbox is reserved for a student project<br />
<Structure load='1ERI' size='500' frame='true' align='right' caption='Insert caption here' scene='Insert optional scene name here' /><br />
== Presentation ==<br />
<br />
<br />
EcoRI is a type II restriction endonuclease. It recognizes and cleaves DNA on a specific palindromic sequence: GAATTC. EcoRI has been extracted from strain R Escherichia coli, a common bacterium which populates the intestine of mammalians. In bacteria, restriction enzymes protect the cell by cutting foreign DNA from bacteriophages (specific bacterial viruses).<br />
<br />
== Reaction ==<br />
<br />
EcoRI (E.C. 3.1.21.4) is a hydrolase and its substrate is a double-strand DNA molecule and two water molecules. For its catalytic activity, EcoRI needs a cofactor which is the divalent ion Mg2+. EcoRI hydrolyses the phosphodiester bond between the guanylic and adenylic residues resulting in 5’-phosphate sticky ends which are complementary.<br />
<br />
== Structure ==<br />
<br />
EcoRI is a homodimer, so it has two identical subunits (<scene name='Sandbox_666/Monomer_structure/3'>monomer strucure shown here</scene>) of 31 kDa, but it possible to have homotetramers at high concentrations.. The constitutive monomers are 276 amino acids long. EcoRI and all the other restriction enzymes show a common structural core which is a α/β domain. The constitutive subunits of EcoRI are organized into a single α/β domain (five stranded β sheet which are surrounded on by α helices). Four of these five β strands are parallel whereas the fourth (β4) is in an anti-parallel orientation to the others.<br />
<br />
The N-terminal section of each subunit forms, with a β-hairpin, an “arm” which wraps around the DNA molecule. (The arm brings the DNA molecule to the catalytic cleft.)<br />
<br />
The specific recognition of EcoRI of the GAATTC sequence is mediated by twelve hydrogen bonds (six bonds per subunit) originating from α helical recognition modules. Three amino acids are responsible for the recognition: Arg200, Glu144 and Arg145, each residue form two hydrogen bonds with Guanine and the adjacent Adenosine residues respectively. <br />
<br />
The reaction is due to a catalytic sequence motif which is found in most type II restriction endonucleases: the PD…D/EXK motif. For EcoRI, this catalytic sequence is PD91 …E111AK.<scene name='Sandbox_666/Catalytic_core/3'>The catalytic core is shown here in red</scene>. This motif is also responsible for Mg2+ binding(Asp90 and Glu111).<br />
<br />
<br />
== Application of EcoRI in molecular biology ==<br />
<br />
Type II restriction endonuclease like EcoRI are often used in molecular biology for their capacity to cut precisely DNA on specific restriction site. That makes them useful tools for gene cloning. By using two different restriction enzymes, it is possible to do directional cloning which is very important if you want to insert a gene in an expression vector.<br />
<br />
<br />
== References ==<br />
<!-- <br />
Please use the "3D" button above this box to insert a Jmol applet (molecule) on this page.<br />
Or use the four-green-boxes-button to insert scrollable text adjacent<br />
to a Jmol applet. Check out the other buttons as well! <br />
--></div>Raphael Bilgerhttps://proteopedia.org/index.php?title=Sandbox_666&diff=1332665Sandbox 6662011-12-15T15:18:08Z<p>Raphael Bilger: </p>
<hr />
<div>Hello this Sandbox is reserved for a student project<br />
<Structure load='1ERI' size='500' frame='true' align='right' caption='Insert caption here' scene='Insert optional scene name here' /><br />
== Presentation ==<br />
<br />
<br />
EcoRI is a type II restriction endonuclease. It recognizes and cleaves DNA on a specific palindromic sequence: GAATTC. EcoRI has been extracted from strain R Escherichia coli, a common bacterium which populates the intestine of mammalians. In bacteria, restriction enzymes protect the cell by cutting foreign DNA from bacteriophages (specific bacterial viruses).<br />
<br />
== Reaction ==<br />
<br />
EcoRI (E.C. 3.1.21.4) is a hydrolase and its substrate is a double-strand DNA molecule and two water molecules. For its catalytic activity, EcoRI needs a cofactor which is the divalent ion Mg2+. EcoRI hydrolyses the phosphodiester bond between the guanylic and adenylic residues resulting in 5’-phosphate sticky ends which are complementary.<br />
<br />
== Structure ==<br />
<br />
EcoRI is a homodimer, so it has two identical subunits (<scene name='Sandbox_666/Monomer_structure/3'>monomer strucure shown here</scene>)of 31 kDa, but it possible to have homotetramers at high concentrations.. The constitutive monomers are 276 amino acids long. EcoRI and all the other restriction enzymes show a common structural core which is a α/β domain. The constitutive subunits of EcoRI are organized into a single α/β domain (five stranded β sheet which are surrounded on by α helices). Four of these five β strands are parallel whereas the fourth (β4) is in an anti-parallel orientation to the others.<br />
<br />
The N-terminal section of each subunit forms, with a β-hairpin, an “arm” which wraps around the DNA molecule. (The arm brings the DNA molecule to the catalytic cleft.)<br />
<br />
The specific recognition of EcoRI of the GAATTC sequence is mediated by twelve hydrogen bonds (six bonds per subunit) originating from α helical recognition modules. Three amino acids are responsible for the recognition: Arg200, Glu144 and Arg145, each residue form two hydrogen bonds with Guanine and the adjacent Adenosine residues respectively. <br />
<br />
The reaction is due to a catalytic sequence motif which is found in most type II restriction endonucleases: the PD…D/EXK motif. For EcoRI, this catalytic sequence is PD91 …E111AK.<scene name='Sandbox_666/Catalytic_core/3'>The catalytic core is shown here in red</scene> motif is also responsible for Mg2+ binding(Asp90 and Glu111).<br />
<br />
<br />
== Application of EcoRI in molecular biology ==<br />
<br />
Type II restriction endonuclease like EcoRI are often used in molecular biology for their capacity to cut precisely DNA on specific restriction site. That makes them useful tools for gene cloning. By using two different restriction enzymes, it is possible to do directional cloning which is very important if you want to insert a gene in an expression vector.<br />
<br />
<br />
== References ==<br />
<!-- <br />
Please use the "3D" button above this box to insert a Jmol applet (molecule) on this page.<br />
Or use the four-green-boxes-button to insert scrollable text adjacent<br />
to a Jmol applet. Check out the other buttons as well! <br />
--></div>Raphael Bilgerhttps://proteopedia.org/index.php?title=Sandbox_666&diff=1332661Sandbox 6662011-12-15T15:01:45Z<p>Raphael Bilger: </p>
<hr />
<div>Hello this Sandbox is reserved for a student project<br />
<Structure load='1ERI' size='500' frame='true' align='right' caption='Insert caption here' scene='Insert optional scene name here' /><br />
== Presentation ==<br />
<br />
<br />
EcoRI is a type II restriction endonuclease. It recognizes and cleaves DNA on a specific palindromic sequence: GAATTC. EcoRI has been extracted from strain R Escherichia coli, a common bacterium which populates the intestine of mammalians. In bacteria, restriction enzymes protect the cell by cutting foreign DNA from bacteriophages (specific bacterial viruses).<br />
<br />
== Reaction ==<br />
<br />
EcoRI (E.C. 3.1.21.4) is a hydrolase and its substrate is a double-strand DNA molecule and two water molecules. For its catalytic activity, EcoRI needs a cofactor which is the divalent ion Mg2+. EcoRI hydrolyses the phosphodiester bond between the guanylic and adenylic residues resulting in 5’-phosphate sticky ends which are complementary.<br />
<br />
== Structure ==<br />
<br />
EcoRI is a homodimer, so it has two identical subunits (<scene name='Sandbox_666/Monomer_structure/3'>monomer strucure shown here</scene>)of 31 kDa, but it possible to have homotetramers at high concentrations.. The constitutive monomers are 276 amino acids long. EcoRI and all the other restriction enzymes show a common structural core which is a α/β domain. The constitutive subunits of EcoRI are organized into a single α/β domain (five stranded β sheet which are surrounded on by α helices). Four of these five β strands are parallel whereas the fourth (β4) is in an anti-parallel orientation to the others.<br />
<br />
The N-terminal section of each subunit forms, with a β-hairpin, an “arm” which wraps around the DNA molecule. (The arm brings the DNA molecule to the catalytic cleft.)<br />
<br />
The specific recognition of EcoRI of the GAATTC sequence is mediated by twelve hydrogen bonds (six bonds per subunit) originating from α helical recognition modules. Three amino acids are responsible for the recognition: Arg200, Glu144 and Arg145, each residue form two hydrogen bonds with Guanine and the adjacent Adenosine residues respectively. <br />
<br />
The reaction is due to a catalytic sequence motif which is found in most type II restriction endonucleases: the PD…D/EXK motif. For EcoRI, this catalytic sequence is PD91 …E111AK.<scene name='Sandbox_666/Catalytic_core/2'>The catalytic core is shown here in red.</scene>This motif is also responsible for Mg2+ binding(Asp90 and Glu111).<br />
<br />
<br />
== Application of EcoRI in molecular biology ==<br />
<br />
Type II restriction endonuclease like EcoRI are often used in molecular biology for their capacity to cut precisely DNA on specific restriction site. That makes them useful tools for gene cloning. By using two different restriction enzymes, it is possible to do directional cloning which is very important if you want to insert a gene in an expression vector.<br />
<br />
<br />
== References ==<br />
<!-- <br />
Please use the "3D" button above this box to insert a Jmol applet (molecule) on this page.<br />
Or use the four-green-boxes-button to insert scrollable text adjacent<br />
to a Jmol applet. Check out the other buttons as well! <br />
--></div>Raphael Bilgerhttps://proteopedia.org/index.php?title=Sandbox_666&diff=1332660Sandbox 6662011-12-15T14:57:34Z<p>Raphael Bilger: </p>
<hr />
<div>Hello this Sandbox is reserved for a student project<br />
<Structure load='1ERI' size='500' frame='true' align='right' caption='Insert caption here' scene='Insert optional scene name here' /><br />
== Presentation ==<br />
<br />
<br />
EcoRI is a type II restriction endonuclease. It recognizes and cleaves DNA on a specific palindromic sequence: GAATTC. EcoRI has been extracted from strain R Escherichia coli, a common bacterium which populates the intestine of mammalians. In bacteria, restriction enzymes protect the cell by cutting foreign DNA from bacteriophages (specific bacterial viruses).<br />
<br />
== Reaction ==<br />
<br />
EcoRI (E.C. 3.1.21.4) is a hydrolase and its substrate is a double-strand DNA molecule and two water molecules. For its catalytic activity, EcoRI needs a cofactor which is the divalent ion Mg2+. EcoRI hydrolyses the phosphodiester bond between the guanylic and adenylic residues resulting in 5’-phosphate sticky ends which are complementary.<br />
<br />
== Structure ==<br />
<br />
EcoRI is a homodimer, so it has two identical subunits (<scene name='Sandbox_666/Monomer_structure/3'>monomer strucure shown here</scene>)of 31 kDa, but it possible to have homotetramers at high concentrations.. The constitutive monomers are 276 amino acids long. EcoRI and all the other restriction enzymes show a common structural core which is a α/β domain. The constitutive subunits of EcoRI are organized into a single α/β domain (five stranded β sheet which are surrounded on by α helices). Four of these five β strands are parallel whereas the fourth (β4) is in an anti-parallel orientation to the others.<br />
<br />
The N-terminal section of each subunit forms, with a β-hairpin, an “arm” which wraps around the DNA molecule. (The arm brings the DNA molecule to the catalytic cleft.)<br />
<br />
The specific recognition of EcoRI of the GAATTC sequence is mediated by twelve hydrogen bonds (six bonds per subunit) originating from α helical recognition modules. Three amino acids are responsible for the recognition: Arg200, Glu144 and Arg145, each residue form two hydrogen bonds with Guanine and the adjacent Adenosine residues respectively. <br />
<br />
The reaction is due to a catalytic sequence motif which is found in most type II restriction endonucleases: the PD…D/EXK motif. For EcoRI, this catalytic sequence is PD91 …E111AK <scene name='Sandbox_666/Catalytic_core/1'>The catalytic core is shown here.</scene> This motif is also responsible for Mg2+ binding(Asp90 and Glu111).<br />
<br />
<br />
== Application of EcoRI in molecular biology ==<br />
<br />
Type II restriction endonuclease like EcoRI are often used in molecular biology for their capacity to cut precisely DNA on specific restriction site. That makes them useful tools for gene cloning. By using two different restriction enzymes, it is possible to do directional cloning which is very important if you want to insert a gene in an expression vector.<br />
<br />
<br />
== References ==<br />
<!-- <br />
Please use the "3D" button above this box to insert a Jmol applet (molecule) on this page.<br />
Or use the four-green-boxes-button to insert scrollable text adjacent<br />
to a Jmol applet. Check out the other buttons as well! <br />
--></div>Raphael Bilgerhttps://proteopedia.org/index.php?title=Sandbox_666&diff=1332659Sandbox 6662011-12-15T14:42:31Z<p>Raphael Bilger: </p>
<hr />
<div>Hello this Sandbox is reserved for a student project<br />
<Structure load='1ERI' size='500' frame='true' align='right' caption='Insert caption here' scene='Insert optional scene name here' /><br />
== Presentation ==<br />
<br />
<br />
EcoRI is a type II restriction endonuclease. It recognizes and cleaves DNA on a specific palindromic sequence: GAATTC. EcoRI has been extracted from strain R Escherichia coli, a common bacterium which populates the intestine of mammalians. In bacteria, restriction enzymes protect the cell by cutting foreign DNA from bacteriophages (specific bacterial viruses).<br />
<br />
== Reaction ==<br />
<br />
EcoRI (E.C. 3.1.21.4) is a hydrolase and its substrate is a double-strand DNA molecule and two water molecules. For its catalytic activity, EcoRI needs a cofactor which is the divalent ion Mg2+. EcoRI hydrolyses the phosphodiester bond between the guanylic and adenylic residues resulting in 5’-phosphate sticky ends which are complementary.<br />
<br />
== Structure ==<br />
<br />
EcoRI is a homodimer, so it has two identical subunits (<scene name='Sandbox_666/Monomer_structure/3'>monomer strucure shown here</scene>)of 31 kDa, but it possible to have homotetramers at high concentrations.. The constitutive monomers are 276 amino acids long. EcoRI and all the other restriction enzymes show a common structural core which is a α/β domain. The constitutive subunits of EcoRI are organized into a single α/β domain (five stranded β sheet which are surrounded on by α helices). Four of these five β strands are parallel whereas the fourth (β4) is in an anti-parallel orientation to the others.<br />
<br />
The N-terminal section of each subunit forms, with a β-hairpin, an “arm” which wraps around the DNA molecule. (The arm brings the DNA molecule to the catalytic cleft.)<br />
<br />
The specific recognition of EcoRI of the GAATTC sequence is mediated by twelve hydrogen bonds (six bonds per subunit) originating from α helical recognition modules. Three amino acids are responsible for the recognition: Arg200, Glu144 and Arg145, each residue form two hydrogen bonds with Guanine and the adjacent Adenosine residues respectively. <br />
<br />
The reaction is due to a catalytic sequence motif which is found in most type II restriction endonucleases: the PD…D/EXK motif. For EcoRI, this catalytic sequence is PD91 …E111AK. This motif is also responsible for Mg2+ binding(Asp90 and Glu111).<br />
<br />
<br />
== Application of EcoRI in molecular biology ==<br />
<br />
Type II restriction endonuclease like EcoRI are often used in molecular biology for their capacity to cut precisely DNA on specific restriction site. That makes them useful tools for gene cloning. By using two different restriction enzymes, it is possible to do directional cloning which is very important if you want to insert a gene in an expression vector.<br />
<br />
<br />
== References ==<br />
<!-- <br />
Please use the "3D" button above this box to insert a Jmol applet (molecule) on this page.<br />
Or use the four-green-boxes-button to insert scrollable text adjacent<br />
to a Jmol applet. Check out the other buttons as well! <br />
--></div>Raphael Bilgerhttps://proteopedia.org/index.php?title=Sandbox_666&diff=1332658Sandbox 6662011-12-15T14:34:22Z<p>Raphael Bilger: </p>
<hr />
<div>Hello this Sandbox is reserved for a student project<br />
<Structure load='1ERI' size='500' frame='true' align='right' caption='Insert caption here' scene='Insert optional scene name here' /><br />
== Presentation ==<br />
<br />
<br />
EcoRI is a type II restriction endonuclease. It recognizes and cleaves DNA on a specific palindromic sequence: GAATTC. EcoRI has been extracted from strain R Escherichia coli, a common bacterium which populates the intestine of mammalians. In bacteria, restriction enzymes protect the cell by cutting foreign DNA from bacteriophages (specific bacterial viruses).<br />
<br />
== Reaction ==<br />
<br />
EcoRI (E.C. 3.1.21.4) is a hydrolase and its substrate is a double-strand DNA molecule and two water molecules. For its catalytic activity, EcoRI needs a cofactor which is the divalent ion Mg2+. EcoRI hydrolyses the phosphodiester bond between the guanylic and adenylic residues resulting in 5’-phosphate sticky ends which are complementary.<br />
<br />
== Structure ==<br />
<br />
EcoRI is a homodimer, so it has two identical subunits (<scene name='Sandbox_666/Monomer_structure/1'>monomer structure</scene>)of 31 kDa, but it possible to have homotetramers at high concentrations.. The constitutive monomers are 276 amino acids long. EcoRI and all the other restriction enzymes show a common structural core which is a α/β domain. The constitutive subunits of EcoRI are organized into a single α/β domain (five stranded β sheet which are surrounded on by α helices). Four of these five β strands are parallel whereas the fourth (β4) is in an anti-parallel orientation to the others.<br />
<br />
The N-terminal section of each subunit forms, with a β-hairpin, an “arm” which wraps around the DNA molecule. (The arm brings the DNA molecule to the catalytic cleft.)<br />
<br />
The specific recognition of EcoRI of the GAATTC sequence is mediated by twelve hydrogen bonds (six bonds per subunit) originating from α helical recognition modules. Three amino acids are responsible for the recognition: Arg200, Glu144 and Arg145, each residue form two hydrogen bonds with Guanine and the adjacent Adenosine residues respectively. <br />
<br />
The reaction is due to a catalytic sequence motif which is found in most type II restriction endonucleases: the PD…D/EXK motif. For EcoRI, this catalytic sequence is PD91 …E111AK. This motif is also responsible for Mg2+ binding(Asp90 and Glu111).<br />
<br />
<br />
== Application of EcoRI in molecular biology ==<br />
<br />
Type II restriction endonuclease like EcoRI are often used in molecular biology for their capacity to cut precisely DNA on specific restriction site. That makes them useful tools for gene cloning. By using two different restriction enzymes, it is possible to do directional cloning which is very important if you want to insert a gene in an expression vector.<br />
<br />
<br />
== References ==<br />
<!-- <br />
Please use the "3D" button above this box to insert a Jmol applet (molecule) on this page.<br />
Or use the four-green-boxes-button to insert scrollable text adjacent<br />
to a Jmol applet. Check out the other buttons as well! <br />
--></div>Raphael Bilgerhttps://proteopedia.org/index.php?title=Sandbox_666&diff=1332657Sandbox 6662011-12-15T14:30:49Z<p>Raphael Bilger: </p>
<hr />
<div>Hello this Sandbox is reserved for a student project<br />
<Structure load='1ERI' size='500' frame='true' align='right' caption='Insert caption here' scene='Insert optional scene name here' /><br />
== Presentation ==<br />
<br />
<br />
EcoRI is a type II restriction endonuclease. It recognizes and cleaves DNA on a specific palindromic sequence: GAATTC. EcoRI has been extracted from strain R Escherichia coli, a common bacterium which populates the intestine of mammalians. In bacteria, restriction enzymes protect the cell by cutting foreign DNA from bacteriophages (specific bacterial viruses).<br />
<br />
== Reaction ==<br />
<br />
EcoRI (E.C. 3.1.21.4) is a hydrolase and its substrate is a double-strand DNA molecule and two water molecules. For its catalytic activity, EcoRI needs a cofactor which is the divalent ion Mg2+. EcoRI hydrolyses the phosphodiester bond between the guanylic and adenylic residues resulting in 5’-phosphate sticky ends which are complementary.<br />
<br />
== Structure ==<br />
<br />
EcoRI is a homodimer, so it has two identical subunits (<scene name='Sandbox_666/Protein_part/1'>monomer structure </scene> of 31 kDa, but it possible to have homotetramers at high concentrations.. The constitutive monomers are 276 amino acids long. EcoRI and all the other restriction enzymes show a common structural core which is a α/β domain. The constitutive subunits of EcoRI are organized into a single α/β domain (five stranded β sheet which are surrounded on by α helices). Four of these five β strands are parallel whereas the fourth (β4) is in an anti-parallel orientation to the others.<br />
<br />
The N-terminal section of each subunit forms, with a β-hairpin, an “arm” which wraps around the DNA molecule. (The arm brings the DNA molecule to the catalytic cleft.)<br />
<br />
The specific recognition of EcoRI of the GAATTC sequence is mediated by twelve hydrogen bonds (six bonds per subunit) originating from α helical recognition modules. Three amino acids are responsible for the recognition: Arg200, Glu144 and Arg145, each residue form two hydrogen bonds with Guanine and the adjacent Adenosine residues respectively. <br />
<br />
The reaction is due to a catalytic sequence motif which is found in most type II restriction endonucleases: the PD…D/EXK motif. For EcoRI, this catalytic sequence is PD91 …E111AK. This motif is also responsible for Mg2+ binding(Asp90 and Glu111).<br />
<br />
<br />
== Application of EcoRI in molecular biology ==<br />
<br />
Type II restriction endonuclease like EcoRI are often used in molecular biology for their capacity to cut precisely DNA on specific restriction site. That makes them useful tools for gene cloning. By using two different restriction enzymes, it is possible to do directional cloning which is very important if you want to insert a gene in an expression vector.<br />
<br />
<br />
== References ==<br />
<!-- <br />
Please use the "3D" button above this box to insert a Jmol applet (molecule) on this page.<br />
Or use the four-green-boxes-button to insert scrollable text adjacent<br />
to a Jmol applet. Check out the other buttons as well! <br />
--></div>Raphael Bilgerhttps://proteopedia.org/index.php?title=Sandbox_666&diff=1332656Sandbox 6662011-12-15T14:16:25Z<p>Raphael Bilger: </p>
<hr />
<div>Hello this Sandbox is reserved for a student project<br />
<Structure load='1ERI' size='500' frame='true' align='right' caption='Insert caption here' scene='Insert optional scene name here' /><br />
== Presentation ==<br />
<br />
<br />
EcoRI is a type II restriction endonuclease. It recognizes and cleaves DNA on a specific palindromic sequence: GAATTC. EcoRI has been extracted from strain R Escherichia coli, a common bacterium which populates the intestine of mammalians. In bacteria, restriction enzymes protect the cell by cutting foreign DNA from bacteriophages (specific bacterial viruses).<br />
<br />
== Reaction ==<br />
<br />
EcoRI (E.C. 3.1.21.4) is a hydrolase and its substrate is a double-strand DNA molecule and two water molecules. For its catalytic activity, EcoRI needs a cofactor which is the divalent ion Mg2+. EcoRI hydrolyses the phosphodiester bond between the guanylic and adenylic residues resulting in 5’-phosphate sticky ends which are complementary.<br />
<br />
== Structure ==<br />
<br />
EcoRI is a homodimer, so it has two identical subunits () of 31 kDa, but it possible to have homotetramers at high concentrations.. The constitutive monomers are 276 amino acids long. EcoRI and all the other restriction enzymes show a common structural core which is a α/β domain. The constitutive subunits of EcoRI are organized into a single α/β domain (five stranded β sheet which are surrounded on by α helices). Four of these five β strands are parallel whereas the fourth (β4) is in an anti-parallel orientation to the others.<br />
<br />
The N-terminal section of each subunit forms, with a β-hairpin, an “arm” which wraps around the DNA molecule. (The arm brings the DNA molecule to the catalytic cleft.)<br />
<br />
The specific recognition of EcoRI of the GAATTC sequence is mediated by twelve hydrogen bonds (six bonds per subunit) originating from α helical recognition modules. Three amino acids are responsible for the recognition: Arg200, Glu144 and Arg145, each residue form two hydrogen bonds with Guanine and the adjacent Adenosine residues respectively. <br />
<br />
The reaction is due to a catalytic sequence motif which is found in most type II restriction endonucleases: the PD…D/EXK motif. For EcoRI, this catalytic sequence is PD91 …E111AK. This motif is also responsible for Mg2+ binding(Asp90 and Glu111).<br />
<br />
<br />
== Application of EcoRI in molecular biology ==<br />
<br />
Type II restriction endonuclease like EcoRI are often used in molecular biology for their capacity to cut precisely DNA on specific restriction site. That makes them useful tools for gene cloning. By using two different restriction enzymes, it is possible to do directional cloning which is very important if you want to insert a gene in an expression vector.<br />
<br />
<br />
== References ==<br />
<!-- <br />
Please use the "3D" button above this box to insert a Jmol applet (molecule) on this page.<br />
Or use the four-green-boxes-button to insert scrollable text adjacent<br />
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--></div>Raphael Bilgerhttps://proteopedia.org/index.php?title=Sandbox_666&diff=1332655Sandbox 6662011-12-15T14:05:50Z<p>Raphael Bilger: </p>
<hr />
<div>Hello this Sandbox is reserved for a student project<br />
<Structure load='1ERI' size='500' frame='true' align='right' caption='Insert caption here' scene='Insert optional scene name here' /><br />
== Presentation ==<br />
<br />
<br />
EcoRI is a type II restriction endonuclease. It recognizes and cleaves DNA on a specific palindromic sequence: GAATTC. EcoRI has been extracted from strain R Escherichia coli, a common bacterium which populates the intestine of mammalians. In bacteria, restriction enzymes protect the cell by cutting foreign DNA from bacteriophages (specific bacterial viruses).<br />
<br />
== Reaction ==<br />
<br />
EcoRI (E.C. 3.1.21.4) is a hydrolase and its substrate is a double-strand DNA molecule and two water molecules. For its catalytic activity, EcoRI needs a cofactor which is the divalent ion Mg2+. EcoRI hydrolyses the phosphodiester bond between the guanylic and adenylic residues resulting in 5’-phosphate sticky ends which are complementary.<br />
<br />
== Structure ==<br />
<br />
EcoRI is a homodimer, so it has two identical subunits (<scene name='Sandbox_666/Monomer/1'>TextToBeDisplayed</scene><scene name='Sandbox_666/Monomer/5'>TextToBeDisplayed</scene>) of 31 kDa, but it possible to have homotetramers at high concentrations.. The constitutive monomers are 276 amino acids long. EcoRI and all the other restriction enzymes show a common structural core which is a α/β domain. The constitutive subunits of EcoRI are organized into a single α/β domain (five stranded β sheet which are surrounded on by α helices). Four of these five β strands are parallel whereas the fourth (β4) is in an anti-parallel orientation to the others.<br />
<br />
The N-terminal section of each subunit forms, with a β-hairpin, an “arm” which wraps around the DNA molecule. (The arm brings the DNA molecule to the catalytic cleft.)<br />
<br />
The specific recognition of EcoRI of the GAATTC sequence is mediated by twelve hydrogen bonds (six bonds per subunit) originating from α helical recognition modules. Three amino acids are responsible for the recognition: Arg200, Glu144 and Arg145, each residue form two hydrogen bonds with Guanine and the adjacent Adenosine residues respectively. <br />
<br />
The reaction is due to a catalytic sequence motif which is found in most type II restriction endonucleases: the PD…D/EXK motif. For EcoRI, this catalytic sequence is PD91 …E111AK. This motif is also responsible for Mg2+ binding(Asp90 and Glu111).<br />
<br />
<br />
== Application of EcoRI in molecular biology ==<br />
<br />
Type II restriction endonuclease like EcoRI are often used in molecular biology for their capacity to cut precisely DNA on specific restriction site. That makes them useful tools for gene cloning. By using two different restriction enzymes, it is possible to do directional cloning which is very important if you want to insert a gene in an expression vector.<br />
<br />
<br />
== References ==<br />
<!-- <br />
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--></div>Raphael Bilgerhttps://proteopedia.org/index.php?title=Sandbox_666&diff=1332654Sandbox 6662011-12-15T14:02:12Z<p>Raphael Bilger: </p>
<hr />
<div>Hello this Sandbox is reserved for a student project<br />
<Structure load='1ERI' size='500' frame='true' align='right' caption='Insert caption here' scene='Insert optional scene name here' /><br />
== Presentation ==<br />
<br />
<br />
EcoRI is a type II restriction endonuclease. It recognizes and cleaves DNA on a specific palindromic sequence: GAATTC. EcoRI has been extracted from strain R Escherichia coli, a common bacterium which populates the intestine of mammalians. In bacteria, restriction enzymes protect the cell by cutting foreign DNA from bacteriophages (specific bacterial viruses).<br />
<br />
== Reaction ==<br />
<br />
EcoRI (E.C. 3.1.21.4) is a hydrolase and its substrate is a double-strand DNA molecule and two water molecules. For its catalytic activity, EcoRI needs a cofactor which is the divalent ion Mg2+. EcoRI hydrolyses the phosphodiester bond between the guanylic and adenylic residues resulting in 5’-phosphate sticky ends which are complementary.<br />
<br />
== Structure ==<br />
<br />
EcoRI is a homodimer, so it has two identical subunits (<scene name='Sandbox_666/Monomer/3'>the constitutive monomer is shown here</scene>) of 31 kDa, but it possible to have homotetramers at high concentrations.. The constitutive monomers are 276 amino acids long. EcoRI and all the other restriction enzymes show a common structural core which is a α/β domain. The constitutive subunits of EcoRI are organized into a single α/β domain (five stranded β sheet which are surrounded on by α helices). Four of these five β strands are parallel whereas the fourth (β4) is in an anti-parallel orientation to the others.<br />
<br />
The N-terminal section of each subunit forms, with a β-hairpin, an “arm” which wraps around the DNA molecule. (The arm brings the DNA molecule to the catalytic cleft.)<br />
<br />
The specific recognition of EcoRI of the GAATTC sequence is mediated by twelve hydrogen bonds (six bonds per subunit) originating from α helical recognition modules. Three amino acids are responsible for the recognition: Arg200, Glu144 and Arg145, each residue form two hydrogen bonds with Guanine and the adjacent Adenosine residues respectively. <br />
<br />
The reaction is due to a catalytic sequence motif which is found in most type II restriction endonucleases: the PD…D/EXK motif. For EcoRI, this catalytic sequence is PD91 …E111AK. This motif is also responsible for Mg2+ binding(Asp90 and Glu111).<br />
<br />
<br />
== Application of EcoRI in molecular biology ==<br />
<br />
Type II restriction endonuclease like EcoRI are often used in molecular biology for their capacity to cut precisely DNA on specific restriction site. That makes them useful tools for gene cloning. By using two different restriction enzymes, it is possible to do directional cloning which is very important if you want to insert a gene in an expression vector.<br />
<br />
<br />
== References ==<br />
<!-- <br />
Please use the "3D" button above this box to insert a Jmol applet (molecule) on this page.<br />
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--></div>Raphael Bilgerhttps://proteopedia.org/index.php?title=Sandbox_666&diff=1332653Sandbox 6662011-12-15T13:49:56Z<p>Raphael Bilger: /* Structure */</p>
<hr />
<div>Hello this Sandbox is reserved for a student project<br />
<Structure load='1ERI' size='500' frame='true' align='right' caption='Insert caption here' scene='Insert optional scene name here' /><br />
== Presentation ==<br />
<br />
<br />
EcoRI is a type II restriction endonuclease. It recognizes and cleaves DNA on a specific palindromic sequence: GAATTC. EcoRI has been extracted from strain R Escherichia coli, a common bacterium which populates the intestine of mammalians. In bacteria, restriction enzymes protect the cell by cutting foreign DNA from bacteriophages (specific bacterial viruses).<br />
<br />
== Reaction ==<br />
<br />
EcoRI (E.C. 3.1.21.4) is a hydrolase and its substrate is a double-strand DNA molecule and two water molecules. For its catalytic activity, EcoRI needs a cofactor which is the divalent ion Mg2+. EcoRI hydrolyses the phosphodiester bond between the guanylic and adenylic residues resulting in 5’-phosphate sticky ends which are complementary.<br />
<br />
== Structure ==<br />
<br />
EcoRI is a homodimer, so it has two identical subunits (<scene name='Sandbox_666/Monomer/3'>the constitutive monomer is shown here</scene>) of 31 kDa, but it possible to have homotetramers at high concentrations.. The constitutive monomers are 276 amino acids long. EcoRI and all the other restriction enzymes show a common structural core which is a α/β domain. The constitutive subunits of EcoRI are organized into a single α/β domain (five stranded β sheet which are surrounded on by α helices). Four of these five β strands are parallel whereas the fourth (β4) is in an anti-parallel orientation to the others.<br />
<br />
The N-terminal section of each subunit forms, with a β-hairpin, an “arm” which wraps around the DNA molecule. (The arm brings the DNA molecule to the catalytic cleft.)<br />
<br />
The specific recognition of EcoRI of the GAATTC sequence is mediated by twelve hydrogen bonds (six bonds per subunit) originating from α helical recognition modules. Three amino acids are responsible for the recognition: Arg200, Glu144 and Arg145, each residue form two hydrogen bonds with Guanine and the adjacent Adenosine residues respectively. <br />
<br />
The reaction is due to a catalytic sequence motif which is found in most type II restriction endonucleases: the PD…D/EXK motif. For EcoRI, this catalytic sequence is PD91 …E111AK. This motif is also responsible for Mg2+ binding(Asp90 and Glu111).<br />
<br />
== References ==<br />
<br />
== Application of EcoRI in molecular biology ==<br />
<br />
Type II restriction endonuclease like EcoRI are often used in molecular biology for their capacity to cut precisely DNA on specific restriction site. That makes them useful tools for gene cloning. By using two different restriction enzymes, it is possible to do directional cloning which is very important if you want to insert a gene in an expression vector.<br />
<!-- <br />
Please use the "3D" button above this box to insert a Jmol applet (molecule) on this page.<br />
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--></div>Raphael Bilgerhttps://proteopedia.org/index.php?title=Sandbox_666&diff=1332652Sandbox 6662011-12-15T13:46:18Z<p>Raphael Bilger: /* Structure */</p>
<hr />
<div>Hello this Sandbox is reserved for a student project<br />
<Structure load='1ERI' size='500' frame='true' align='right' caption='Insert caption here' scene='Insert optional scene name here' /><br />
== Presentation ==<br />
<br />
<br />
EcoRI is a type II restriction endonuclease. It recognizes and cleaves DNA on a specific palindromic sequence: GAATTC. EcoRI has been extracted from strain R Escherichia coli, a common bacterium which populates the intestine of mammalians. In bacteria, restriction enzymes protect the cell by cutting foreign DNA from bacteriophages (specific bacterial viruses).<br />
<br />
== Reaction ==<br />
<br />
EcoRI (E.C. 3.1.21.4) is a hydrolase and its substrate is a double-strand DNA molecule and two water molecules. For its catalytic activity, EcoRI needs a cofactor which is the divalent ion Mg2+. EcoRI hydrolyses the phosphodiester bond between the guanylic and adenylic residues resulting in 5’-phosphate sticky ends which are complementary.<br />
<br />
== Structure ==<br />
<br />
EcoRI is a homodimer, so it has two identical subunits (<scene name='Sandbox_666/Monomer/3'>the constitutive monomer is shown here</scene>) of 31 kDa, but it possible to have homotetramers at high concentrations.. The constitutive monomers are 276 amino acids long. EcoRI and all the other restriction enzymes show a common structural core which is a α/β domain. The constitutive subunits of EcoRI are organized into a single α/β domain (five stranded β sheet which are surrounded on by α helices). Four β strands are parallel whereas the fourth is in an anti-parallel orientation to the others.<br />
<br />
The N-terminal section of each subunit forms, with a β-hairpin, an “arm” which wraps around the DNA molecule. (The arm brings the DNA molecule to the catalytic cleft.)<br />
<br />
The specific recognition of EcoRI of the GAATTC sequence is mediated by twelve hydrogen bonds (six bonds per subunit) originating from α helical recognition modules. Three amino acids are responsible for the recognition: Arg200, Glu144 and Arg145, each residue form two hydrogen bonds with Guanine and the adjacent Adenosine residues respectively. <br />
<br />
The reaction is due to a catalytic sequence motif which is found in most type II restriction endonucleases: the PD…D/EXK motif. For EcoRI, this catalytic sequence is PD91 …E111AK. This motif is also responsible for Mg2+ binding(Asp90 and Glu111).<br />
<br />
== Application of EcoRI in molecular biology ==<br />
<br />
Type II restriction endonuclease like EcoRI are often used in molecular biology for their capacity to cut precisely DNA on specific restriction site. That makes them useful tools for gene cloning. By using two different restriction enzymes, it is possible to do directional cloning which is very important if you want to insert a gene in an expression vector.<br />
<!-- <br />
Please use the "3D" button above this box to insert a Jmol applet (molecule) on this page.<br />
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--></div>Raphael Bilger