engleski

Maintaining the protein biosynthesis fidelity by isoleucyl-tRNA synthetase

Aminoacyl-tRNA synthetases (aaRS) supply the protein biosynthesis machinery with aminoacyl- tRNAs, activated precursors of the amino acids incorporated in the growing polypeptide chains on the ribosomes. Any erroneous amino acid activation by aaRS compromises the fidelity of translation as no additional surveillance mechanisms of charged tRNAs exist, and the erroneous amino acid will be misincorporated into the nascent polypeptide chains. Isoleucyl-tRNA synthetase (IleRS) is challenged to discriminate two closely related isoleucine mimics: valine and norvaline. Both amino acids are smaller than isoleucine by having one methyl group less, and neatly fit in the active site of IleRS. Valine is a naturally occurring proteinogenic amino acid omnipresent in living cells, while norvaline (Nva) is a nonproteinogenic metabolite present in bacterial cells only under special circumstances, such as during the anaerobic growth conditions. We have explored the mechanisms and differences in Val and Nva misactivation and proofreading by IleRS, and the consequences of mistranslation of Ile codons by Val and Nva in vivo. As expected, Nva and Val are activated in the active site of IleRS and efficiently transferred to tRNA. Therefore, IleRS possesses additional editing domain with hydrolytic activity, to cleave off noncognate Nva or Val transferred to tRNA. The hydrolysis of Nva- tRNAIle is faster than Val- tRNAIle cleavage, so misacylation of tRNAIle with Nva proceeds at slower rate than Val tRNAIle misacylation. Accordingly, an Escherichia coli strain lacking IleRS editing activity misincorporated Nva and Val in the proteome to a similar extent. However, Nva mistranslation resulted in higher toxicity than Val, in agreement with IleRS editing being optimized for Nva-tRNAIle clearance. It noteworthy that evolutionary related leucyl- and valyl-tRNA synthetases also efficiently hydrolyse Nva-tRNAs, pointing out to possibility that Nva editing is of ancient origin and already present in the common ancestor of the IleRS, LeuRS and ValRS, preventing the norvaline infiltration into the proteins since the early stages of evolution.

isoleucyl-tRNA synthetase ; translation ; editing ; tRNA

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