Sandbox 201: Difference between revisions
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'''T4 RNA ligase 1 (Rnl1)''' | |||
---- | |||
{{STRUCTURE_2c5u|right| PDB=2C5U | SIZE=300 | SCENE=}} | {{STRUCTURE_2c5u|right| PDB=2C5U | SIZE=300 | SCENE=}} | ||
[[Image:Overall_structure_of_T4_RNA_ligase.png|thumb|left|An overall structure of the T4 RNA ligase (Rnl1) with AMPcPP. Alpha-helices are colored in ''cyan''. The beta-strands are colored in ''red''. Loops are colored in ''purple''. The AMPcPP molecule is shown as a stick drawing in ''yellow''.]] | [[Image:Overall_structure_of_T4_RNA_ligase.png|thumb|left|An overall structure of the T4 RNA ligase (Rnl1) with AMPcPP. Alpha-helices are colored in ''cyan''. The beta-strands are colored in ''red''. Loops are colored in ''purple''. The AMPcPP molecule is shown as a stick drawing in ''yellow''.]] | ||
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Rnl1 is in fact a tRNA repair enzyme used by the T4 bacteriophage to escape hosts antiviral response. Enzyme functioning requires ATP and divalent metal ions. The T4 ligase repairs the tRNA<sup>Lys</sup> by joining its 5'-PO<sub>4</sub> and 3'-OH groups via series of three nucleotidyl transfer steps in a ping-pong enzymatic mechanism. First, the Lys<sup>99</sup> of the enzyme reacts with the a phosphorus of ATP and forms a covalent intermediate: ligase-(lysyl-N)-AMP. Pyrophosphate is also produced during this step. Secondly, AMP is transferred from the intermediate to the 5'- PO<sub>4</sub> terminus of a tRNA to form an tRNA-adenylate intermediate (AppRNA). Finally, the ligase catalyzes the attack of the 3'-OH terminus of the tRNA on the tRNA-adenylate and the two termini are joined via a phosphodiester bond, the AMP is released. | Rnl1 is in fact a tRNA repair enzyme used by the T4 bacteriophage to escape hosts antiviral response. Enzyme functioning requires ATP and divalent metal ions. The T4 ligase repairs the tRNA<sup>Lys</sup> by joining its 5'-PO<sub>4</sub> and 3'-OH groups via series of three nucleotidyl transfer steps in a ping-pong enzymatic mechanism. First, the Lys<sup>99</sup> of the enzyme reacts with the a phosphorus of ATP and forms a covalent intermediate: ligase-(lysyl-N)-AMP. Pyrophosphate is also produced during this step. Secondly, AMP is transferred from the intermediate to the 5'- PO<sub>4</sub> terminus of a tRNA to form an tRNA-adenylate intermediate (AppRNA). Finally, the ligase catalyzes the attack of the 3'-OH terminus of the tRNA on the tRNA-adenylate and the two termini are joined via a phosphodiester bond, the AMP is released. | ||
<br/><br/> | <br/><br/><br/> | ||
==Biological role== | ==Biological role== | ||
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:The enzyme binds <scene name='Sandbox_201/Ca/1'>four calcium ions</scene> Ca<sup>2+</sup>. <scene name='Sandbox_201/Ca6_apc_residues/2'>Two</scene> are coordinated to six water molecules. They do not directly interact with the enzyme, but via water molecules interacting with Glu227, Glu159, Lys99, Glu100, and Tyr246 via hydrogen bonds. They also interact with one phosphoryl oxygen of the AMPcPP. <scene name='Sandbox_201/Ca4_residues/3'>Two other</scene> are coordinated to four water molecules and interact with three enzyme residues (Ile211 and Asp212) via hydrogen bonds. | :The enzyme binds <scene name='Sandbox_201/Ca/1'>four calcium ions</scene> Ca<sup>2+</sup>. <scene name='Sandbox_201/Ca6_apc_residues/2'>Two</scene> are coordinated to six water molecules. They do not directly interact with the enzyme, but via water molecules interacting with Glu227, Glu159, Lys99, Glu100, and Tyr246 via hydrogen bonds. They also interact with one phosphoryl oxygen of the AMPcPP. <scene name='Sandbox_201/Ca4_residues/3'>Two other</scene> are coordinated to four water molecules and interact with three enzyme residues (Ile211 and Asp212) via hydrogen bonds. | ||
:Calcium is very important for enzyme Rnl1 structural biology, because the enzyme crystallizes only in présence of Ca<sup>2+</sup>. This could be explained by the fact that interactions between Ca<sup>2+</sup> and negatively charged surface of each Rnl1 allow interactions between several enzymes at crystallization interfaces.<ref name="main_article" /> | :Calcium is very important for enzyme Rnl1 structural biology, because the enzyme crystallizes only in présence of Ca<sup>2+</sup>. This could be explained by the fact that interactions between Ca<sup>2+</sup> and negatively charged surface of each Rnl1 allow interactions between several enzymes at crystallization interfaces.<ref name="main_article" /> | ||
* Anion binding sites | |||
:The enzyme binds <scene name='Sandbox_201/Cl/1'>three chloride ions</scene> Cl<sup>-</sup>. | |||
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::- The RNA have to be close to the ATP binding site to enable the AMP transfer from Lys99. The RNA could bind at the surface of the C-terminal domain, allowing the anticodon loop to be positionned toward the ATP binding site. | ::- The RNA have to be close to the ATP binding site to enable the AMP transfer from Lys99. The RNA could bind at the surface of the C-terminal domain, allowing the anticodon loop to be positionned toward the ATP binding site. | ||
::- Moreover, the C-terminal helical structure matches the tRNA structure. | ::- Moreover, the C-terminal helical structure matches the tRNA structure. | ||
:The Rnl1 C-terminal domain is unique in nucleotidyltransferase family, so the architecture of this domain could allow T4 RNA ligase to bind specifically tRNA<sup>Lys</sup> ''in vivo''. | |||
:A chloride ion is also positionned in the active site, and could mimic the 5'-phosphate of the incoming RNA.<ref>Odell, M., Malinina, L., Sriskanda, V., Teplova, M., and Shuman, S. (2003) Nucleic Acids Res. 31, 5090–5100</ref><ref>Odell, M., Sriskanda, V., Shuman, S., and Nikolov, D. B. (2000) Mol. Cell 6, 1183–1193</ref><ref name="main_article" /> | |||
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</StructureSection> | </StructureSection> | ||
<br/><br/> | |||
==Chemical reaction== | |||
:The T4 RNA ligase (Enzyme class : E.C.6.5.1.3) catalyzes the formation of phosphodiester bonds between the 5'-phosphate terminus of single-stranded nucleic acid (i) and the 3'-hydroxyl terminus of single-stranded nucleic acid (j). | |||
:'''ATP + ribonucleotide<sub>(i)</sub> + ribonucleotide<sub>(j)</sub> → AMP + diphosphate + ribonucleotide<sub>(i+j)</sub>''' | |||
= | :Rnl1 catalysis involves three steps : | ||
::* In the first step, Lys99 in the conserved motif KX(D/N)G (motif I) reacts with the α-phosphate of ATP or ATP analogue (NAD or GTP) and forms a covalent bond. This step gives a covalent intermediate ligase-(lysyl-N)-AMP and a pyrophosphate. Lys99, which is responsible for this step, is the essential residue involved in the catalytic mechanism.<ref>Shuman, S., and Schwer, B. (1995) Mol. Microbiol. 17, 405–410</ref> | |||
::* In the second step, AMP is transferred from the covalent intermediate to the 5'-phosphate RNA, forming a tRNA-adenylate intermediate (AppRNA). Arg54, by stabilizing and orienting the RNA phosphate by hydrogen bonding, is essential for this RNA adenylation.<ref name="main_article" /> | |||
::* In the third step, the 3'-hydroxyl RNA attacks the 5'-phosphate RNA. A phosphodiester bond is formed and an AMP is released. | |||
==See Also== | |||
*[[T4 RNA ligase 2 (Rnl2)]] | |||
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*[http://www.ebi.ac.uk/pdbsum/2c5u PDBsum file on 2C5U] | *[http://www.ebi.ac.uk/pdbsum/2c5u PDBsum file on 2C5U] | ||
*[http://oca.weizmann.ac.il/oca-bin/ocaids?id=2c5u OCA Atlas for 2C5U] | *[http://oca.weizmann.ac.il/oca-bin/ocaids?id=2c5u OCA Atlas for 2C5U] | ||
==References== | ==References== | ||
<references /> | <references /> | ||
