Sandbox 201: Difference between revisions
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T4 RNA ligase (Rnl1) catalyzes the formation of phosphodiester bonds between the 5'-phosphate and the 3'-hydroxyl termini of single-stranded nucleic acids. | T4 RNA ligase (Rnl1) catalyzes the formation of phosphodiester bonds between the 5'-phosphate and the 3'-hydroxyl termini of single-stranded nucleic acids. | ||
T4 RNA ligase is a member of a distinct subgroup of RNA ligases along with a fungal [[TRNA|tRNA]] ligase(Trl1), a putative baculovirus RNA ligase and RNA ligase from the bacteriophages RM378 and TS2126. Rnl1 is also the first RNA ligase whose complete crystal structure was determined. | T4 RNA ligase is a member of a distinct subgroup of RNA ligases along with a fungal [[TRNA|tRNA]] ligase (Trl1), a putative baculovirus RNA ligase and RNA ligase from the bacteriophages RM378 and TS2126. Rnl1 is also the first RNA ligase whose complete crystal structure was determined. | ||
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. | ||
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* <scene name='Sandbox_201/Atp_binding_site_grey/1'>ATP binding site</scene> | * <scene name='Sandbox_201/Atp_binding_site_grey/1'>ATP binding site</scene> | ||
:The N- and C-terminal domains are both able to form interactions with ATP and ATP analogues. But the ß-strands of the core region in the N-terminal domain contain most of the residues involved in binding ATP. <scene name='Sandbox_201/Lys75_green_ball_v1/ | :The N- and C-terminal domains are both able to form interactions with ATP and ATP analogues. But the ß-strands of the core region in the N-terminal domain contain most of the residues involved in binding ATP. <scene name='Sandbox_201/Lys75_green_ball_v1/3'>Lys75</scene>, <scene name='Sandbox_201/Atp_binding_site_lys99/1'>Lys99</scene> (motif I), <scene name='Sandbox_201/Atp_binding_site_lys119/1'>Lys119</scene> (motif Ia), <scene name='Sandbox_201/Atp_binding_site_lys240/1'>Lys240</scene> (motif V), and <scene name='Sandbox_201/Atp_binding_site_lys242/1'>Lys242</scene> (motif V) interact with the phosphate groups of ATP and ATP analogues. | ||
:Lys99 is the site of adenylation in Rnl1, <ref>Thogersen, H. C., Morris, H. R., Rand, K. N., and Gait, M. J. (1985) Eur. J. Biochem. | :Lys99 is the site of adenylation in Rnl1, <ref>Thogersen, H. C., Morris, H. R., Rand, K. N., and Gait, M. J. (1985) Eur. J. Biochem. | ||
147, 325–329</ref> but in this structure this residue seems to be situated at a distance incompatible with covalent interaction with the phosphate of ATP (more than 3 Å).<ref name="main_article">K.El Omari, J.Ren, L.E.Bird, M.K.Bona, G.Klarmann, S.F.LeGrice, D.K.Stammers (2006) J. Biol. Chem. 281,1573-1579</ref> That could suggest that the formation of a covalent bond needs some conformational changes. But we do not know if a conformational change has to occur to allow the covalent bond formation, or if the formation of this bond leads to a conformational change. | 147, 325–329</ref> but in this structure this residue seems to be situated at a distance incompatible with covalent interaction with the phosphate of ATP (more than 3 Å).<ref name="main_article">K.El Omari, J.Ren, L.E.Bird, M.K.Bona, G.Klarmann, S.F.LeGrice, D.K.Stammers (2006) J. Biol. Chem. 281,1573-1579</ref> That could suggest that the formation of a covalent bond needs some conformational changes. But we do not know if a conformational change has to occur to allow the covalent bond formation, or if the formation of this bond leads to a conformational change. | ||
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==Chemical reaction== | ==Chemical reaction== | ||
The T4 RNA ligase 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). | 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> | ATP + ribonucleotide<sub>(i)</sub> + ribonucleotide<sub>(j)</sub> → AMP + diphosphate + ribonucleotide<sub>(i+j)</sub> | ||
