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

Nevena Cvetesic,Mirna Bilus,Ita Gruic-Sovulj

doi:10.1074/jbc.m115.648568

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.

Highlights

Isoleucyl-tRNA synthetase uses cognate tRNA to stimulate hydrolysis of non-cognate aminoacyl-adenylates within the synthetic site
The IleRS1⁄7tRNAIle complex is probably a superior catalyst under cellular conditions compared with isoleucyl-tRNA synthetase (IleRS) alone because (i) its affinity for amino acids is better adjusted to the cellular environment, and (ii) only in the case of valine is the tRNA-dependent rate of hydrolysis comparable with the rate of the aminoacyl transfer step, and contributes to proofreading
The Intact 3Ј-End Is Not Required for the Participation of tRNA in Pre-transfer Editing—IleRS exhibits inherent hydrolytic activity toward aminoacyl-AMP, which can be substantially modulated by the tRNA (Table 1) [12, 28]

Summary

Background

Isoleucyl-tRNA synthetase uses cognate tRNA to stimulate hydrolysis of non-cognate aminoacyl-adenylates within the synthetic site. 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 pretransfer editing within the IleRS synthetic site and further demonstrates that the A76 hydroxyl groups participate in posttransfer editing only. We further demonstrate that the tRNA cofactor does not facilitate discrimination between isoleucine and valine in either of these reactions Despite these findings, the IleRS1⁄7tRNAIle complex is probably a superior catalyst under cellular conditions compared with IleRS alone because (i) its affinity for amino acids is better adjusted to the cellular environment, and (ii) only in the case of valine is the tRNA-dependent rate of hydrolysis comparable with the rate of the aminoacyl transfer step, and contributes to proofreading. The separation of the pre- and post-transfer editing pathways by various tRNAIle analogues clearly demonstrated that tRNA participates in IleRS proofreading by two distinct mechanisms; (i) the intact 3Ј-end of tRNA permits post-transfer editing by supporting the misaminoacylation/deacylation pathway, whereas (ii) the remaining portions of the tRNA body promote a conformational change that stimulates pre-transfer editing within the IleRS synthetic site

Experimental Procedures

Results

31 Ϯ 1 31

Discussion