Mechanisms have evolved to prevent errors in replication, transcription, and translation of genetic material, with translational errors occurring most frequently. Errors in protein synthesis can ...occur at two steps, during tRNA aminoacylation and ribosome decoding. Recent advances in protein mass spectrometry have indicated that previous reports of translational errors have potentially underestimated the frequency of these events, but also that the majority of translational errors occur during ribosomal decoding, suggesting that aminoacylation errors are evolutionarily less tolerated. Despite that interpretation, there is evidence that some aminoacylation errors may be regulated, and thus provide a benefit to the cell, while others are clearly detrimental. Here, we show that while it has been suggested that regulated Thr-to-Ser substitutions may be beneficial, there is a threshold beyond which these errors are detrimental. In contrast, we show that errors mediated by alanyl-tRNA synthetase (AlaRS) are not well tolerated and induce a global stress response that leads to gross perturbation of the
proteome, with potentially catastrophic effects on fitness and viability. Tolerance for Ala mistranslation appears to be much lower than with other translational errors, consistent with previous reports of multiple proofreading mechanisms targeting mischarged tRNA
These results demonstrate the essential role of aminoacyl-tRNA proofreading in optimizing cellular fitness and suggest that any potentially beneficial effects of mistranslation may be confined to specific amino acid substitutions.
Errors in protein synthesis have historically been assumed to be detrimental to the cell. While there are many reports that translational errors are consequential, there is a growing body of evidence that some mistranslation events may be tolerated or even beneficial. Using two models of mistranslation, we compare the direct phenotypic effects of these events in
This work provides insight into the threshold for tolerance of specific mistranslation events that were previously predicted to be broadly neutral to proteome integrity. Furthermore, these data reveal the effects of mistranslation beyond the general unfolded stress response, leading to global translational reprogramming.
One integral step in the transition from a nucleic acid encoded-genome to functional proteins is the aminoacylation of tRNA molecules. To perform this activity, aminoacyl-tRNA synthetases (aaRSs) ...activate free amino acids in the cell forming an aminoacyl-adenylate before transferring the amino acid on to its cognate tRNA. These newly formed aminoacyl-tRNA (aa-tRNA) can then be used by the ribosome during mRNA decoding. In Escherichia coli, there are twenty aaRSs encoded in the genome, each of which corresponds to one of the twenty proteinogenic amino acids used in translation. Given the shared chemicophysical properties of many amino acids, aaRSs have evolved mechanisms to prevent erroneous aa-tRNA formation with non-cognate amino acid substrates. Of particular interest is the post-transfer proofreading activity of alanyl-tRNA synthetase (AlaRS) which prevents the accumulation of Ser-tRNAAla and Gly-tRNAAla in the cell. We have previously shown that defects in AlaRS proofreading of Ser-tRNAAla lead to global dysregulation of the E. coli proteome, subsequently causing defects in growth, motility, and antibiotic sensitivity. Here we report second-site AlaRS suppressor mutations that alleviate the aforementioned phenotypes, revealing previously uncharacterized residues within the AlaRS proofreading domain that function in quality control.
Aminoacyl-tRNA synthetases (aaRSs) are enzymes that synthesize aminoacyl-tRNAs to facilitate translation of the genetic code. Quality control by aaRS proofreading and other mechanisms maintains ...translational accuracy, which promotes cellular viability. Systematic disruption of proofreading, as recently demonstrated for alanyl-tRNA synthetase (AlaRS), leads to dysregulation of the proteome and reduced viability. Recent studies showed that environmental challenges such as exposure to reactive oxygen species can also alter aaRS synthetic and proofreading functions, prompting us to investigate if oxidation might positively or negatively affect AlaRS activity. We found that while oxidation leads to modification of several residues in Escherichia coli AlaRS, unlike in other aaRSs, this does not affect proofreading activity against the noncognate substrates serine and glycine and only results in a 1.6-fold decrease in efficiency of cognate Ala-tRNAAla formation. Mass spectrometry analysis of oxidized AlaRS revealed that the critical proofreading residue in the editing site, Cys666, and three methionine residues (M217 in the active site, M658 in the editing site, and M785 in the C-Ala domain) were modified to cysteine sulfenic acid and methionine sulfoxide, respectively. Alanine scanning mutagenesis showed that none of the identified residues were solely responsible for the change in cognate tRNAAla aminoacylation observed under oxidative stress, suggesting that these residues may act as reactive oxygen species “sinks” to protect catalytically critical sites from oxidative damage. Combined, our results indicate that E. coli AlaRS proofreading is resistant to oxidative damage, providing an important mechanism of stress resistance that helps to maintain proteome integrity and cellular viability.
Accuracy during translation ensures faithful conversion of the genetic code. Aminoacyl tRNA-synthetases (aaRS) are essential enzymes that attach an amino acid to a cognate tRNA. The aaRS first ...activates an amino acid in an ATP-dependent manner to form an aminoacyl adenylate. Next, the aaRS attaches the aminoacyl adenylate to the 3’-OH of the terminal adenosine residue of the cognate tRNA. The aminoacylated tRNA is transported to the ribosome by the elongation factor Tu and GTP to participate in translation. AaRSs play a major role in maintaining translational accuracy by attaching the correct amino acids to their cognate tRNAs. Although the active site pockets of many aaRSs are able to discriminate against the wrong amino acid, the shared chemicophysical structure of some amino acids can lead to mis-activated amino acids. To maintain translation accuracy, aaRS have evolved proofreading strategies that can hydrolyze a mis-activated aminoacyl adenylate in the active site or translocate a misacylated tRNA to a separate editing domain capable of hydrolysis. The proofreading strategies ensure fidelity during aminoacylation and prevent mistranslation. It has been previously shown that disruption of the editing domain of alanyl-tRNA synthetase (AlaRS) in mice causes neurodegeneration. In E. coli, perturbation to the editing domain through mutagenesis of a critical cysteine residue, Cys666, leads to the global dysregulation of the E. coli proteome resulting in a wide range of phenotypes including growth defects, impaired motility, and antibiotic sensitivity.Here we show that the stress caused by the replacement of the critical cysteine in the editing domain of E. coli AlaRS selects for spontaneous second-site suppressor mutations that alter the editing domain to alleviate the phenotypic defects. This work highlights the importance of maintaining AlaRS fidelity during aminoacylation and characterizes novel features of the AlaRS editing domain.Recent studies showed that environmental challenges such as exposure to reactive oxygen species (ROS) can also alter aaRS synthetic and proofreading functions, prompting us to investigate if oxidation might positively or negatively affect AlaRS activity. Here we show that oxidative stress does not affect the proofreading function of E. coli AlaRS but leads to a decrease in the rate of tRNAAla aminoacylation. Additionally, residues that undergo oxidative modification were identified using mass spectrometry and their contribution to the regulation of E. coli AlaRS under oxidative stress were analyzed. This study indicates that E. coli AlaRS proofreading is resistant to oxidative damage, providing an important mechanism of stress resistance that helps to maintain proteome integrity and cellular viability.
To ensure that correct amino acids are incorporated during protein synthesis, aminoacyl‐tRNA synthetases (aaRSs) use proofreading mechanisms collectively referred to as editing. Although editing is ...important for viability, editing‐deficient aaRSs have been identified in host‐dependent organisms. In Mycoplasma mobile, editing‐deficient PheRS and LeuRS have been identified. We characterized the amino acid activation site of MmPheRS and identified a previously unknown hyperaccurate mutation, L287F. Additionally, we report that m‐Tyr, an oxidation byproduct of Phe which is toxic to editing‐deficient cells, is poorly discriminated by MmPheRS activation and is not subjected to editing. Furthermore, expressing MmPheRS and the hyperaccurate variants renders Escherichia coli susceptible to m‐Tyr stress, indicating that active site discrimination is insufficient in tolerating excess m‐Tyr.
This study examined the amino acid activation center of the Mycoplasma mobile aminoacyl‐tRNA synthetase PheRS through point mutation and enzyme kinetics. A trend of m‐Tyr (an oxidation byproduct of Phe) being poorly discriminated during the step of activation was observed. Since MmPheRS does not edit m‐Tyr, these findings suggest that M. mobile may be susceptible to m‐Tyr toxicity, when Phe is limited unless other quality control mechanisms exist.
Abstract
Aminoacyl‐tRNA synthetases (aaRSs) maintain translational fidelity by ensuring the formation of correct aminoacyl‐tRNA pairs. Numerous point mutations in human aaRSs have been linked to ...disease phenotypes. Structural studies of aaRSs from human pathogens encoding unique domains support these enzymes as potential candidates for therapeutics. Studies have shown that the identity of tRNA pools in cells changes between different cell types and under stress conditions. While traditional radioactive aminoacylation analyses can determine the effect of disease‐causing mutations on aaRS function, these assays are not amenable to drug discovery campaigns and do not take into account the variability of the intracellular tRNA pools. Here, we review modern techniques to characterize aaRS activity
in vitro
and in cells. The cell‐based approaches analyse the aminoacyl‐tRNA pool to observe trends in aaRS activity and fidelity. Taken together, these approaches allow high‐throughput drug screening of aaRS inhibitors and systems‐level analyses of the dynamic tRNA population under a variety of conditions and disease states.
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