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Detecting mistranslation from a serine tRNA with proline anticodon expressed in yeast

ABSTRACT: Mistranslation, the mis-incorporation of an amino acid not specified by the “standard” genetic code, occurs in all cells. tRNA variants that increase mistranslation arise spontaneously and engineered tRNAs can achieve mistranslation frequencies approaching 10% in yeast and bacteria. The goal of this study was to detect and quantify mistranslation from a serine tRNA variant with proline UGG anticodon and G26A secondary mutation engineered in yeast

INSTRUMENT(S): Orbitrap Exploris 480

ORGANISM(S): Saccharomyces Cerevisiae (baker's Yeast)

SUBMITTER: Matthew Berg

LAB HEAD: Judit Villen

PROVIDER: PXD032063 | Pride | 2022-08-12

REPOSITORIES: Pride

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Genetic background and mistranslation frequency determine the impact of mistranslating tRNASerUGG.

Berg Matthew D MD Zhu Yanrui Y Loll-Krippleber Raphaël R San Luis Bryan-Joseph BJ Genereaux Julie J Boone Charles C Villén Judit J Brown Grant W GW Brandl Christopher J CJ

G3 (Bethesda, Md.) 20220701 7

Transfer RNA variants increase the frequency of mistranslation, the misincorporation of an amino acid not specified by the "standard" genetic code, to frequencies approaching 10% in yeast and bacteria. Cells cope with these variants by having multiple copies of each tRNA isodecoder and through pathways that deal with proteotoxic stress. In this study, we define the genetic interactions of the gene encoding tRNASerUGG,G26A, which mistranslates serine at proline codons. Using a collection of yeast ...[more]

PMID: 35587152

Similar Datasets

Detecting mistranslation in yeast expressing tRNA variants

Project description:Mistranslation, the mis-incorporation of an amino acid not specified by the “standard” genetic code, occurs in all cells. tRNA variants that increase mistranslation arise spontaneously and engineered tRNAs can achieve mistranslation frequencies approaching 10% in yeast and bacteria. The goal of this study was to detect mistranslation from two different tRNA variants. The first variant, tRNA-Pro-G3:U70, has a mutation in its acceptor stem creating a G3:U70 base pair which is the key identity element for the alanine tRNA synthetase. This tRNA should be charged with alanine and mis-incorporate alanine at proline codons. The second variant, tRNA-Ser-UCU,G26A, is a serine tRNA with an arginine anticodon and a G26A secondary mutation to dampen function and prevent lethal levels of mistranslation. This tRNA should mis-incorporate serine at arginine codons.

2021-05-17 | PXD025934 | Pride

Transcriptome profile of S. cerevisiae strains expressing mistranslating tRNAs

2021-05-11 | GSE174145 | GEO

Quantification of proline to serine mistranslation in a Saccharomyces cerevisiae strain expressing an inducible tRNA variant

Project description:Altering the genetic code for applications in synthetic biology and genetic code expansion involves engineered tRNAs that incorporate amino acids that differ from what is defined by the “standard” genetic code. Since these engineered tRNA variants can be lethal due to proteotoxic stress, regulating their expression is necessary to achieve high levels of the resulting novel proteins. Mechanisms to positively regulate transcription with exogenous activator proteins like those often used to regulate RNA polymerase II (RNAP II) transcribed genes are not applicable to tRNAs as their expression by RNA polymerase III requires elements internal to the tRNA. Here, we show that tRNA expression is repressed by overlapping transcription from an adjacent RNAP II promoter. Regulating the expression of the RNAP II promoter allows inverse regulation of the tRNA. Placing either Gal4 or TetR-VP16 activated promoters downstream of a mistranslating tRNA serine variant that mis-incorporates serine at proline codons in Saccharomyces cerevisiae allows mistranslation at a level not otherwise possible because of the toxicity of the unregulated tRNA. Using mass spectrometry, we determine th frequency of mistranslation in both the induced and repressed conditions of the galactose inducible and tetracycline inducible systems.

2021-11-03 | PXD028496 | Pride

Identification of proline to serine mistranslation in Drosophila melanogaster pupae

Project description:Transfer RNAs (tRNAs) are the adaptor molecules required for reading of the genetic code and the accurate production of proteins. tRNA variants can lead to genome-wide mistranslation, the misincorporation of amino acids not specified by the standard genetic code, into nascent proteins. While genome sequencing has identified putative mistranslating tRNA variants in human populations, little is known regarding how mistranslation affects multicellular organisms. Here, we create a Drosophila melanogaster model for mistranslation by integrating a serine tRNA variant that mistranslates serine for proline (tRNA(Ser)[UGG, G26A]) into the fly genome. Using mass spectrometry, we find that tRNA(Ser)[UGG, G26A] misincorporates serine for proline at a frequency of ~ 0.6% per codon. This model will enable studies into the synergistic effects of mistranslating tRNA variants and disease-causing alleles.

2021-09-18 | PXD028498 | Pride

Transcription profiling of yeast in response to mRNA mistranslation after 20 hours of induction

Project description:Transcriptome analysis in cells after 20 hours of mistranslation induction in relation to control cells at the same time point where mistranslation was not induced (without the inducible tRNA construction)

2010-10-01 | E-MTAB-165 | biostudies-arrayexpress

Identification of nascent peptides of peptidyl-tRNA dissociated from ribosome in the early elongation stage in Escherichia coli pthts cells

Project description:Accurate translation of mRNAs into functional proteins is a fundamental process in all living organisms. In bacteria, in the early stage of translation elongation, peptidyl-tRNAs (pep-tRNAs) with short nascent chains frequently dissociate from the ribosome (pep-tRNA drop-off). The dissociated pep-tRNAs are deacylated and recycled by peptidyl-tRNA hydrolase (PTH), which is an essential enzyme in bacteria. Here, we establish a highly sensitive method for direct profiling of pep-tRNAs using RNA isolation method and mass spectrometry. We isolated each tRNA species with peptide from Escherichia coli pthts cells using reciprocal circulating chromatography and precisely analyzed their nascent peptides. As a result, we successfully detected 703 peptides consisted of 402 cognate peptides and 301 non-cognate peptides with single amino-acid substitution. Detailed analysis of individual pep-tRNAs revealed that most of the substitutions in the miscoded peptides take place at the C-terminal drop-off site. We further examined this observation using a reporter construct and found that the non-cognate pep-tRNAs produced by mistranslation rarely participate in the next round of elongation but dissociate from the ribosome, suggesting that pep-tRNA drop-off is an active mechanism by which the ribosome rejects miscoded pep-tRNAs in the early elongation, thereby contributing to quality control of protein synthesis after peptide bond formation.

2023-05-18 | PXD030808 | Pride

Identification of nascent peptides of peptidyl-tRNA dissociated from ribosome in the early elongation stage in Escherichia coli pthts cells

2023-05-18 | PXD030807 | Pride

Quantifying mistranslation from alanine tRNA anticodon variants in S. cerevisiae

Project description:Transfer RNAs (tRNAs) maintain translational fidelity through strict charging by their cognate aminoacyl-tRNA synthetase and codon:anticodon base pairing with the mRNA at the ribosome. Mistranslation occurs when an amino acid not specified by the genetic code is incorporated into a protein. Since alanyl-tRNA synthetase uniquely recognizes a G3:U70 base pair in alanine tRNAs and the anticodon plays no role in charging, alanine tRNA variants with anticodon mutations have the potential to mistranslate alanine. Our goal was to quantify mis-incorporation of alanine into proteins in Saccharomyces cerevisiae strains expressing one of 57 different alanine tRNA anticodon variants. Using mass spectrometry, we observed mistranslation for 45 of the variants when expressed on single-copy plasmids.

2023-10-02 | PXD038242 | Pride

Identification of nascent peptides of peptidyl-tRNA dissociated from ribosome in the early elongation stage in Escherichia coli pthts cells

2023-05-18 | PXD030806 | Pride

Identification of nascent peptides of peptidyl-tRNA dissociated from ribosome in the early elongation stage in Escherichia coli pthts cells

2023-05-18 | PXD030805 | Pride