The tRNA A76 Hydroxyl Groups Control Partitioning of the tRNA-dependent Pre- and Post-transfer Editing Pathways in Class I tRNA Synthetase

• Published in: The Journal of biological chemistry 2015-05, Vol.290 (22), p.13981-13991
• Main Authors: Cvetesic, Nevena, Bilus, Mirna, Gruic-Sovulj, Ita
• Format: Article
• Language: English
• Subjects: Adenosine Triphosphate - chemistry; Amino Acids - chemistry; Amino Acyl-tRNA Synthetases - chemistry; aminoacyl-tRNA synthetase; Binding Sites; Catalysis; Escherichia coli - enzymology; Hydrolysis; Isoleucine - chemistry; Isoleucine-tRNA Ligase - chemistry; Isoleucine-tRNA Ligase - genetics; isoleucyl-tRNA synthetase; Phosphates - chemistry; proofreading; Protein Conformation; protein synthesis; Protein Synthesis and Degradation; protein-nucleic acid interaction; ribonuclear protein (RNP); RNA Editing; RNA Precursors - chemistry; RNA, Transfer - chemistry; RNA-Binding Proteins - chemistry; Substrate Specificity; transfer RNA (tRNA); tRNA-dependent pre-transfer editing; Valine - chemistry
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M115.648568

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 tRNAIle organizes the synthetic site to enhance pre-transfer editing, an idiosyncratic feature of IleRS, remains elusive. Here we show that tRNAIle 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-tRNAIle synthesis under cellular conditions. Finally, the extent to which tRNAIle 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. Background: Isoleucyl-tRNA synthetase uses cognate tRNA to stimulate hydrolysis of non-cognate aminoacyl-adenylates within the synthetic site. Results: The 3′-terminal hydroxyl groups of tRNAIle have no role in pre-transfer editing. Conclusion: The tRNAIle body, rather than the 3′-end of tRNAIle alone, promotes assembly of the improved ribonuclear protein synthetic site. Significance: Isoleucyl-tRNA synthetase acts as a ribonuclear protein to adjust amino acid recognition to the cellular environment.