- Award ID(s):
- 1715840
- NSF-PAR ID:
- 10163494
- Date Published:
- Journal Name:
- mBio
- Volume:
- 10
- Issue:
- 6
- ISSN:
- 2150-7511
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
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.more » « less
-
null (Ed.)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.more » « less
-
β- N -methylamino- l -alanine (BMAA) is a nonproteinogenic amino acid that has been associated with neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) and Alzheimer's disease (AD). BMAA has been found in human protein extracts; however, the mechanism by which it enters the proteome is still unclear. It has been suggested that BMAA is misincorporated at serine codons during protein synthesis, but direct evidence of its cotranslational incorporation is currently lacking. Here, using LC-MS–purified BMAA and several biochemical assays, we sought to determine whether any aminoacyl-tRNA synthetase (aaRS) utilizes BMAA as a substrate for aminoacylation. Despite BMAA's previously predicted misincorporation at serine codons, following a screen for amino acid activation in ATP/PP i exchange assays, we observed that BMAA is not a substrate for human seryl-tRNA synthetase (SerRS). Instead, we observed that BMAA is a substrate for human alanyl-tRNA synthetase (AlaRS) and can form BMAA-tRNA Ala by escaping from the intrinsic AlaRS proofreading activity. Furthermore, we found that BMAA inhibits both the cognate amino acid activation and the editing functions of AlaRS. Our results reveal that, in addition to being misincorporated during translation, BMAA may be able to disrupt the integrity of protein synthesis through multiple different mechanisms.more » « less
-
Abstract In Escherichia coli, inconsistencies between in vitro tRNA aminoacylation measurements and in vivo protein synthesis demands were postulated almost 40 years ago, but have proven difficult to confirm. Whole-cell modeling can test whether a cell behaves in a physiologically correct manner when parameterized with in vitro measurements by providing a holistic representation of cellular processes in vivo. Here, a mechanistic model of tRNA aminoacylation, codon-based polypeptide elongation, and N-terminal methionine cleavage was incorporated into a developing whole-cell model of E. coli. Subsequent analysis confirmed the insufficiency of aminoacyl-tRNA synthetase kinetic measurements for cellular proteome maintenance, and estimated aminoacyl-tRNA synthetase kcats that were on average 7.6-fold higher. Simulating cell growth with perturbed kcats demonstrated the global impact of these in vitro measurements on cellular phenotypes. For example, an insufficient kcat for HisRS caused protein synthesis to be less robust to the natural variability in aminoacyl-tRNA synthetase expression in single cells. More surprisingly, insufficient ArgRS activity led to catastrophic impacts on arginine biosynthesis due to underexpressed N-acetylglutamate synthase, where translation depends on repeated CGG codons. Overall, the expanded E. coli model deepens understanding of how translation operates in an in vivo context.
-
Abstract has been an important model for determining the frequency of translational misreading events, those in which a tRNA pairs incorrectly to the mRNA and inserts an amino acid not specified by the codon in the mRNA. Misreading errors have been quantified in vivo using reporter protein systems or mass spectrometry with both approaches converging on a simple model for most misreading. The available data show that misreading tRNAs must form stereotypical base mismatches that correspond to those that can mimic Watson–Crick base pairs when formed in the ribosomal A site. Errors involving other mismatches occur significantly less frequently. This work debunks the idea of an average misreading frequency of 5 × 10−4per codon that extends across the genetic code. Instead, errors come in two distinct classes—high frequency and low frequency events—with most errors being of the low frequency type. A comparison of misreading errors inSaccharomyces cerevisiae andS. cerevisiae suggests the existence of a mechanism that reduces misreading frequency in yeast; this mechanism may operate in eukaryotes generally.Escherichia coli