Year: 2016
Type: article
Abstract: Accurate pairing of amino acids and tRNAs by aminoacyl-tRNA synthetases is a primary checkpoint in maintaining the fidelity of translation of the genetic code.Misacylated aa-tRNAs utilized by the ribosome specify insertion of amino acids into protein at positions not defined by the genetic code.As such, multiple mechanisms have evolved to limit the production of misacylated aa-tRNAs, including strict discrimination against inappropriate amino acids and tRNA that may compete for an aaRS and hydrolysis of the products of erroneous aa-tRNA synthesis.Recently, many environmental conditions that temporarily increase the frequency of aa-tRNA mischarging as well as organisms with unusually high error rates in translation have been discovered, suggesting both that the fidelity of translation of the genetic code is a fluid system and that the requirements for strict quality control in translation are more relaxed in some distinct environmental contexts than in others.Following the observation that tyrosine starvation results in accumulation of phenylalanine at Tyr codons in protein produced Chinese hamster ovary cell culture, we examine quality control by tyrosyl-tRNA synthetase (TyrRS).We find that, under normal growth conditions, discrimination against Phe in the amino acid activation step is sufficient to limit Phe-tRNA Tyr , but under conditions of Tyr starvation, the relative iii abundance of Tyr:Phe drops significantly, resulting in accumulation of Phe-tRNA Tyr species and mistranslation of Tyr codons as Phe.We characterize mutant variants of CHO TyrRS and address the divergence between bacterial and eukaryotic substrate specificity determinants in TyrRS.Bacterial phenylalanyl-tRNA synthetase (PheRS) bears a proofreading domain that catalyzes the hydrolysis of misacylated aa-tRNA Phe species in an active site distinct from the synthetic core.Under normal conditions, the activity of this post-transfer editing domain is dispensable.Like CHO TyrRS, bacterial PheRS displays strict discrimination against most non-cognates in the amino acid activation step.We performed extensive phenotypic and kinetic characterization of Escherichia coli PheRS, and find that posttransfer editing is evolutionarily conserved to protect the cell against cytotoxic mistranslation of Phe codons arising from misacylation of tRNA Phe species with the nonprotein amino acid meta-tyrosine, which accumulates under oxidative stress.m-Tyr is one of many oxidized derivatives of the normal PheRS substrate, Phe.PheRS in E. coli therefore limits mistranslation by both strong discrimination against most non-cognate amino acids in the activation step, as well as post-transfer editing of a non-protein amino acid that accumulates in a specific environmental stress condition.Saccharomyces cerevisiae cytoplasmic PheRS, unlike EcPheRS, does not discriminate against non-cognate amino acids efficiently in the activation step.Instead, it relies on post-transfer editing activity to limit production of misacylated tRNA Phe species with non-cognate amino acids that would otherwise not be efficiently activated by the bacterial enzyme.Given the difference between EcPheRS and SccytoPheRS in the iv mechanisms that limit production of misacylated aa-tRNA Phe species, we performed more extensive analysis of the repertoire of non-cognate amino acids that threaten the selectivity of SccytoPheRS.We find that this enzyme bears at least three distinct mechanisms to limit production of misacylated aa-tRNA Phe , particularly those species derived from non-protein products of oxidative Phe damage.Unexpectedly, defective post-transfer editing is discovered to play a non-canonical role in regulation of cell growth under conditions of amino acid stress.v DEDICATION This work is dedicated to my families of both genetics and circumstance.
Author: Adil Moghal
Institution: The Ohio State University
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W2598136571