Transcriptome profile of S. cerevisiae strains expressing mistranslating tRNAs
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ABSTRACT:
ORGANISM(S): Saccharomyces cerevisiae
PROVIDER: GSE174145 | GEO | 2021/05/11
REPOSITORIES: GEO
Similar Datasets
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
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 and quantify mistranslation from a serine tRNA variant with proline UGG anticodon and G26A secondary mutation engineered in yeast
2022-08-12 | PXD032063 | Pride
Project description:By physically linking amino acids to their codon assignments, transfer RNAs (tRNAs) are essential for protein synthesis and translation fidelity. Some natural human tRNA variants cause amino acid mis-incorporation at a codon or set of codons. Recent work showed a naturally occurring tRNASer variant decodes phenylalanine codons with serine and inhibits protein synthesis. We hypothesized that human tRNA variants that mis-read glycine (Gly) codons with alanine (Ala) will disrupt protein homeostasis. The A3G mutation occurs naturally in tRNAGly variants (tRNAGlyCCC, tRNAGlyGCC) and creates an alanyl-tRNA synthetase (AlaRS) identity element (G3:U70). Because AlaRS does not recognize the anticodon, the human tRNAAlaAGC G35C (tRNAAlaACC) variant may function similarly to mis-incorporate Ala at Gly codons. The tRNAGly and tRNAAla variants had no effect on protein synthesis in mammalian cells under normal growth conditions, however, tRNAGlyGCC A3G depressed protein synthesis in the context of proteasome inhibition. Mass spectrometry confirmed Ala mistranslation at several Gly codons caused by the tRNAGlyGCC A3G and tRNAAlaAGC G35C mutants, and in some cases, we observed multiple mistranslation events in the same peptide. The data reveal mistranslation of Ala at Gly codons and defects in protein homeostasis generated by natural human tRNA variants that are tolerated under normal conditions.
2024-05-21 | PXD034180 | Pride
Project description:“Inaccurate” expression of the genetic code, also known as mistranslation, is an emerging paradigm in microbial studies. Growing evidence indicates that many microbial pathogens can deliberately mistranslate their genetic code to help invade a host or evade host immune responses. However, discovering new capacities for deliberate mistranslation remains a challenge because each group of pathogens typically employs a unique mistranslation mechanism. In this study, we address this problem by studying duplicated genes of aminoacyl-tRNA synthetases. Using bacterial prolyl-tRNA synthe¬tase (ProRS) genes as an example, we identify an anomalous ProRS isoform, ProRSx, and a corre¬sponding tRNA, tRNAProA, that are predominately found in plant pathogens from Streptomyces species. We then show that tRNAProA has an unusual hybrid structure that allows this tRNA to mistranslate alanine codons as proline. We finally provide biochemical, genetic, and mass-spectro-metric evidence that cells which express ProRSx and tRNAProA can translate GCU alanine codons both as alanine and proline. This dual use of alanine codons creates a hidden proteome diversity due to stochastic Ala→Pro mutations in protein sequences. Thus, we show that important plant pathogens are equipped with a tool to alter the identity of their sense codons. This finding reveals the first example of a natural tRNA synthetase/tRNA pair for dedicated mistranslation of sense codons.
2022-02-22 | PXD026636 | Pride
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
Project description:To investigate how organisms mitigate the deleterious effects of mistranslation during evolution, a mutant tRNA was expressed in S. cerevisiae. The expression of Candida Ser-tRNACAG from a low copy plasmid in S. cerevisiae promoted mistranslation events by random incorporation of both serine and leucine at CUG codons. As mistranslation causes an overload of the protein quality pathways, it disrupts cellular protein homeostasis leading to a major fall in fitness. Laboratory evolutionary experiments were performed to study whether the fitness cost of mistranslation can be lowered. We also wanted to identify the cost-reduction strategy: reducing the frequencies of errors (mitigation), or increasing tolerance to errors (robustness), either by global or local activities. Gene expression was measured in the ancestor (non-evolved) lineage in two different situations: 1) carrying an empty vector and 2) expressing the mutant Ser-tRNACAG. Gene expression was also measured in three ambiguously evolved lineages (B3, D11, H9) in both situations (carrying an empty vector and expressing the mutant tRNA). Three independent experiments were performed for each lineage. Non-evolved strain with empty vector was used as control sample.
2015-11-06 | E-GEOD-65718 | biostudies-arrayexpress
Project description:To investigate how organisms mitigate the deleterious effects of mistranslation during evolution, a mutant tRNA was expressed in S. cerevisiae. The expression of Candida Ser-tRNACAG from a low copy plasmid in S. cerevisiae promoted mistranslation events by random incorporation of both serine and leucine at CUG codons. As mistranslation causes an overload of the protein quality pathways, it disrupts cellular protein homeostasis leading to a major fall in fitness. Laboratory evolutionary experiments were performed to study whether the fitness cost of mistranslation can be lowered. We also wanted to identify the cost-reduction strategy: reducing the frequencies of errors (mitigation), or increasing tolerance to errors (robustness), either by global or local activities.
2015-11-06 | GSE65718 | GEO
Project description:Recent works show that protein mistranslation is widespread in nature and that both single cell or multicellular organisms can take advantage of it, by regulating its levels, under specific physiological and environmental conditions. In C. albicans, it leads to increased morphological and physiological phenotypic diversity of high adaptive potential, but the scope of such protein mistranslation is poorly understood due to technical difficulties in detecting and quantifying amino acid misincorporation events in complex proteomic samples. The phenomenon of mistranslation has been extensively studied in the leucine CUG codon, which has been reassigned to either serine or alanine, or ambiguously assigned to serine and leucine in several fungal species of the so called CTG clade. Serine-to-leucine (Ser→Leu) ambiguous decoding has been observed in Ascoidea asiatica, Candida maltosa, C. albicans and more recently, in the halotolerant yeast Debaryomyces hansenii. In C. albicans, CUG translation is facilitated by a hybrid tRNA(CAG)Ser that contains identity elements for both seryl-tRNA synthetase (SerRS) and leucyl-tRNA synthetase (LeuRS). Under normal physiological conditions the tRNA(CAG)Ser is mainly aminoacylated with Ser by the SerRS (appx 97%). We have developed and optimized mass spectrometry and bioinformatics pipelines capable of identifying low-level amino acid misincorporation events at the proteome level and determine codons error frequencies. We have also analysed the proteomic profile of an engineered C. albicans strain that exhibits high level of leucine misincorporation at protein CUG sites.
2024-09-09 | PXD047025 | Pride
Project description:Transfer RNAs (tRNAs) are vital in determining the specificity of translation. Mutations in tRNAs can result in the mis-incorporation of amino acids into nascent polypeptides in a process known as mistranslation. Here, our goal was to test the impacts of different types of mistranslation in the model organism Drosophila melanogaster, as impact of mistranslation depends on the type of amino acid substitution. We created two fly lines - one expressing a serine tRNA variant with valine anticodon and the other with a serine tRNA variant with a threonine anticodon. Using mass spectrometry, we measure the amount of mistranslation at various points in fly development.
2024-06-15 | PXD052492 | Pride
Project description:The genetic code is the cellular translation table for the conversion of nucleotide sequences into amino acid sequences. Changes to the meaning of sense codons would introduce errors into almost every translated message and are expected to be highly detrimental. However, reassignment of single or multiple codons in mitochondria and nuclear genomes, although extremely rare, demonstrates that the code can evolve. Several models for the mechanism of alteration of nuclear genetic codes have been proposed (including ‘codon capture’, ‘genome streamlining’ and ‘ambiguous intermediate’ theories), but with little resolution. Here, we report a novel sense codon reassignment in Pachysolen tannophilus, a yeast related to the Pichiaceae. By generating proteomics data and using tRNA sequence comparisons we show that Pachysolen translates CUG codons as alanine and not as the more usual leucine. The Pachysolen tRNA(CAG) is an anticodon-mutated Ala-tRNA containing all major alanine tRNA recognition sites. The polyphyly of the CUG-decoding tRNAs in yeasts is best explained by a tRNA loss driven codon reassignment mechanism. Loss of the CUG-tRNA in the ancient yeast is followed by gradual decrease of respective codons and subsequent codon capture by tRNAs whose anticodon is not part of the aminoacyl-tRNA synthetase recognition region.
2016-05-06 | PXD003898 | Pride