title: The tRNA A76 Hydroxyl Groups Control Partitioning of the tRNA-dependent Pre- and Post-transfer Editing Pathways in Class I tRNA Synthetase.
abstract: Aminoacyl-tRNA synthetases catalyze ATP-dependent covalent coupling of cognate amino acids and tRNAs for ribosomal protein synthesis. Escherichia coli isoleucyl-tRNA synthetase (IleRS) exploits both the tRNA-dependent pre- and post-transfer editing pathways to minimize errors in translation. However, the molecular mechanisms by which tRNA(Ile) organizes the synthetic site to enhance pre-transfer editing, an idiosyncratic feature of IleRS, remains elusive. Here we show that tRNA(Ile) affects both the synthetic and editing reactions localized within the IleRS synthetic site. In a complex with cognate tRNA, IleRS exhibits a 10-fold faster aminoacyl-AMP hydrolysis and a 10-fold drop in amino acid affinity relative to the free enzyme. Remarkably, the specificity against non-cognate valine was not improved by the presence of tRNA in either of these processes. Instead, amino acid specificity is determined by the protein component per se, whereas the tRNA promotes catalytic performance of the synthetic site, bringing about less error-prone and kinetically optimized isoleucyl-tRNA(Ile) synthesis under cellular conditions. Finally, the extent to which tRNA(Ile) modulates activation and pre-transfer editing is independent of the intactness of its 3'-end. This finding decouples aminoacylation and pre-transfer editing within the IleRS synthetic site and further demonstrates that the A76 hydroxyl groups participate in post-transfer editing only. The data are consistent with a model whereby the 3'-end of the tRNA remains free to sample different positions within the IleRS·tRNA complex, whereas the fine-tuning of the synthetic site is attained via conformational rearrangement of the enzyme through the interactions with the remaining parts of the tRNA body.
authors: From the Department of Chemistry, University of Zagreb, Faculty of Science, Horvatovac 102a, 10000 Zagreb, Croatia.
authors: Nevena
authors: N
authors: Cvetesic
authors: From the Department of Chemistry, University of Zagreb, Faculty of Science, Horvatovac 102a, 10000 Zagreb, Croatia.
authors: Mirna
authors: M
authors: Bilus
authors: From the Department of Chemistry, University of Zagreb, Faculty of Science, Horvatovac 102a, 10000 Zagreb, Croatia gruic@chem.pmf.hr.
authors: Ita
authors: I
authors: Gruic-Sovulj
mesh_terms: D000255:Adenosine Triphosphate / Q000737:chemistry; D000596:Amino Acids / Q000737:chemistry; D000604:Amino Acyl-tRNA Synthetases / Q000737:chemistry*; D001665:Binding Sites; D002384:Catalysis; D004926:Escherichia coli / Q000201:enzymology; D006868:Hydrolysis; D007532:Isoleucine / Q000737:chemistry; D007533:Isoleucine-tRNA Ligase / Q000737:chemistry / Q000235:genetics*; D010710:Phosphates / Q000737:chemistry; D011487:Protein Conformation; D017393:RNA Editing; D012322:RNA Precursors / Q000737:chemistry*; D012343:RNA, Transfer / Q000737:chemistry*; D016601:RNA-Binding Proteins / Q000737:chemistry; D013379:Substrate Specificity; D014633:Valine / Q000737:chemistry
chemical_list: D000596:Amino Acids; D010710:Phosphates; D012322:RNA Precursors; D016601:RNA-Binding Proteins; D007532:Isoleucine; D000255:Adenosine Triphosphate; D012343:RNA, Transfer; D000604:Amino Acyl-tRNA Synthetases; D007533:Isoleucine-tRNA Ligase; D014633:Valine
keywords: aminoacyl-tRNA synthetase; isoleucyl-tRNA synthetase; proofreading; protein synthesis; protein-nucleic acid interaction; ribonuclear protein (RNP); tRNA-dependent pre-transfer editing; transfer RNA (tRNA)