Accurate translation of the genetic code is maintained in part by aminoacyl-tRNA synthetases (aaRS) proofreading mechanisms that ensure correct attachment of a cognate amino acid to a transfer RNA (tRNA). During environmental stress, such as oxidative stress, demands on aaRS proofreading are altered by changes in the availability of cytoplasmic amino acids. For example, oxidative stress increases levels of cytotoxic tyrosine isomers, noncognate amino acids normally excluded from translation by the proofreading activity of phenylalanyl-tRNA synthetase (PheRS). Here we show that oxidation of PheRS induces a conformational change, generating a partially unstructured protein. This conformational change does not affect Phe or Tyr activation or the aminoacylation activity of PheRS. However, in vitro and ex vivo analyses reveal that proofreading activity to hydrolyze Tyr-tRNA Phe is increased during oxidative stress, while the cognate Phe-tRNA Phe aminoacylation activity is unchanged. In HPX − , Escherichia coli that lack reactive oxygen-scavenging enzymes and accumulate intracellular H 2 O 2 , we found that PheRS proofreading is increased by 11%, thereby providing potential protection against hazardous cytoplasmic m -Tyr accumulation. These findings show that in response to oxidative stress, PheRS proofreading is positively regulated without negative effects on the enzyme’s housekeeping activity in translation. Our findings also illustrate that while the loss of quality control and mistranslation may be beneficial under some conditions, increased proofreading provides a mechanism for the cell to appropriately respond to environmental changes during oxidative stress.
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Fine-Tuning of Alanyl-tRNA Synthetase Quality Control Alleviates Global Dysregulation of the Proteome
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.
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- Award ID(s):
- 2101998
- NSF-PAR ID:
- 10232729
- Date Published:
- Journal Name:
- Genes
- Volume:
- 11
- Issue:
- 10
- ISSN:
- 2073-4425
- Page Range / eLocation ID:
- 1222
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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ABSTRACT 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 Escherichia coli 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 Ala . 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. IMPORTANCE 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 Escherichia coli . 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.more » « less
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Abstract In this comprehensive review, I focus on the twenty
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- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.3390/genes11101222
