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Partitioning of tRNA-dependent editing between pre- and post-transfer pathways in class I aminoacyl-tRNA synthetases.

ABSTRACT: Hydrolytic editing activities are present in aminoacyl-tRNA synthetases possessing reduced amino acid discrimination in the synthetic reactions. Post-transfer hydrolysis of misacylated tRNA in class I editing enzymes occurs in a spatially separate domain inserted into the catalytic Rossmann fold, but the location and mechanisms of pre-transfer hydrolysis of misactivated amino acids have been uncertain. Here, we use novel kinetic approaches to distinguish among three models for pre-transfer editing by Escherichia coli isoleucyl-tRNA synthetase (IleRS). We demonstrate that tRNA-dependent hydrolysis of noncognate valyl-adenylate by IleRS is largely insensitive to mutations in the editing domain of the enzyme and that noncatalytic hydrolysis after release is too slow to account for the observed rate of clearing. Measurements of the microscopic rate constants for amino acid transfer to tRNA in IleRS and the related valyl-tRNA synthetase (ValRS) further suggest that pre-transfer editing in IleRS is an enzyme-catalyzed activity residing in the synthetic active site. In this model, the balance between pre-transfer and post-transfer editing pathways is controlled by kinetic partitioning of the noncognate aminoacyl-adenylate. Rate constants for hydrolysis and transfer of a noncognate intermediate are roughly equal in IleRS, whereas in ValRS transfer to tRNA is 200-fold faster than hydrolysis. In consequence, editing by ValRS occurs nearly exclusively by post-transfer hydrolysis in the editing domain, whereas in IleRS both pre- and post-transfer editing are important. In both enzymes, the rates of amino acid transfer to tRNA are similar for cognate and noncognate aminoacyl-adenylates, providing a significant contrast with editing DNA polymerases.

SUBMITTER: Dulic M

PROVIDER: S-EPMC2911327 | biostudies-literature | 2010 Jul

REPOSITORIES: biostudies-literature

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Partitioning of tRNA-dependent editing between pre- and post-transfer pathways in class I aminoacyl-tRNA synthetases.

Dulic Morana M Cvetesic Nevena N Perona John J JJ Gruic-Sovulj Ita I

The Journal of biological chemistry 20100524 31

Hydrolytic editing activities are present in aminoacyl-tRNA synthetases possessing reduced amino acid discrimination in the synthetic reactions. Post-transfer hydrolysis of misacylated tRNA in class I editing enzymes occurs in a spatially separate domain inserted into the catalytic Rossmann fold, but the location and mechanisms of pre-transfer hydrolysis of misactivated amino acids have been uncertain. Here, we use novel kinetic approaches to distinguish among three models for pre-transfer editi ...[more]

PMID: 20498377

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Project description:UnlabelledAminoacyl-tRNA synthetases provide the first step in protein synthesis quality control by discriminating cognate from noncognate amino acid and tRNA substrates. While substrate specificity is enhanced in many instances by cis- and trans-editing pathways, it has been revealed that in organisms such as Streptococcus pneumoniae some aminoacyl-tRNA synthetases display significant tRNA mischarging activity. To investigate the extent of tRNA mischarging in this pathogen, the aminoacylation profiles of class I isoleucyl-tRNA synthetase (IleRS) and class II lysyl-tRNA synthetase (LysRS) were determined. Pneumococcal IleRS mischarged tRNA(Ile) with both Val, as demonstrated in other bacteria, and Leu in a tRNA sequence-dependent manner. IleRS substrate specificity was achieved in an editing-independent manner, indicating that tRNA mischarging would only be significant under growth conditions where Ile is depleted. Pneumococcal LysRS was found to misaminoacylate tRNA(Lys) with Ala and to a lesser extent Thr and Ser, with mischarging efficiency modulated by the presence of an unusual U4:G69 wobble pair in the acceptor stems of both pneumococcal tRNA(Lys) isoacceptors. Addition of the trans-editing factor MurM, which also functions in peptidoglycan synthesis, reduced Ala-tRNA(Lys) production by LysRS, providing evidence for cross talk between the protein synthesis and cell wall biogenesis pathways. Mischarging of tRNA(Lys) by AlaRS was also observed, and this would provide additional potential MurM substrates. More broadly, the extensive mischarging activities now described for a number of Streptococcus pneumoniae aminoacyl-tRNA synthetases suggest that adaptive misaminoacylation may contribute significantly to the viability of this pathogen during amino acid starvation.ImportanceStreptococcus pneumoniae is a common causative agent of several debilitating and potentially life-threatening infections, such as pneumonia, meningitis, and infectious endocarditis. Such infections are increasingly difficult to treat due to widespread development of penicillin resistance. High-level penicillin resistance is known to depend in part upon MurM, a protein involved in both aminoacyl-tRNA-dependent synthesis of indirect amino acid cross-linkages within cell wall peptidoglycan and in translation quality control. The involvement of MurM in both protein synthesis and antibiotic resistance identify it as a potential target for the development of new and potent antibiotics for pneumococcal infections. The goals of this work were to identify and characterize S. pneumoniae pathways that can synthesize mischarged tRNAs and to relate these activities to expected changes in protein and peptidoglycan biosynthesis during antibiotic and nutritional stress.

Dataset Information

Partitioning of tRNA-dependent editing between pre- and post-transfer pathways in class I aminoacyl-tRNA synthetases.

Publications

Partitioning of tRNA-dependent editing between pre- and post-transfer pathways in class I aminoacyl-tRNA synthetases.

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