Modulating Mistranslation Potential of tRNASer in<i>Saccharomyces cerevisiae</i>

Berg, Matthew D.; Zhu, Yi; Genereaux, Julie; Ruiz, Bianca Y.; Rodriguez‐Mias, Ricard A.; Tyler, Allan; Bahcheli, Alexander T; Villén, Judit; Brandl, Christopher J.

doi:10.1534/genetics.119.302525

Cited by 25 publications

(97 citation statements)

References 90 publications

(126 reference statements)

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“…This allows the construction of tRNAs that insert these amino acids at non-cognate codons. Many of these tRNAs have been engineered (for example see Geslain et al 2010;Zimmerman et al 2018;Berg et al 2019). Through analysis similar to that performed here, these mistranslating tRNAs will allow the identification of functionally significant missense mutations in alanine, serine and leucine codons.…”

Section: Discussionmentioning

confidence: 90%

“…In contrast to the concerns addressed by Crick in his "Frozen Accident Theory" (Crick 1968), mistranslation is not catastrophic for cell viability. At levels in the 3-5% range mistranslation has minimal affect on yeast cell viability Lant et al 2017;Berg et al 2019). For some key cellular proteins, 3-5% mistranslation is sufficient to restore viability.…”

Section: Discussionmentioning

confidence: 99%

“…Previously, we engineered serine tRNA variants that mis-incorporate serine at proline codons by replacing the UGA anticodon with UGG (Berg et al , 2019. These tRNAs contain secondary mutations to reduce tRNA functionality and modulate mistranslation levels since plasmids expressing a tRNA with this anticodon change alone can not be transformed into yeast.…”

Section: Introductionmentioning

confidence: 99%

“…These tRNAs contain secondary mutations to reduce tRNA functionality and modulate mistranslation levels since plasmids expressing a tRNA with this anticodon change alone can not be transformed into yeast. One variant, tRNA Ser UGG, G26A, contains a G26A secondary mutation and when expressed from a centromeric plasmid results in a frequency of serine incorporation at proline codons of ~ 5% as determined by mass spectrometry (Berg et al 2019). Zimmerman et al (2018) and Geslain et al (2010) have also demonstrated the possibility of mistranslating serine for a number of amino acids in yeast and mammalian cells.…”

Section: Introductionmentioning

confidence: 99%

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Mistranslating tRNA identifies a deleterious S213P mutation in theSaccharomyces cerevisiae eco1-1allele

Zhu

Berg

Yang

et al. 2020

Preprint

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Mistranslation occurs when an amino acid not specified by the standard genetic code is incorporated during translation. Since the ribosome does not read the amino acid, tRNA variants aminoacylated with a non-cognate amino acid or containing a non-cognate anticodon dramatically increase the frequency of mistranslation. In a systematic genetic analysis, we identified a suppression interaction between tRNA Ser UGG, G26A, which mistranslates proline codons by inserting serine, and eco1-1, a temperature sensitive allele of the gene encoding an acetyltransferase required for sister chromatid cohesion. The suppression was partial with a tRNA that inserts alanine at proline codons and not apparent for a tRNA that inserts serine at arginine codons. Sequencing of the eco1-1 allele revealed a mutation that would convert the highly conserved serine 213 within β7 of the GCN5-related N-acetyltransferase core to proline. Mutation of P213 in eco1-1 back to the wild-type serine restored function of the enzyme at elevated temperature. Our results indicate the utility of mistranslating tRNA variants to identify functionally relevant mutations and identify eco1 as a reporter for mistranslation. We propose that mistranslation could be used as a tool to treat genetic disease.

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Section: Discussionmentioning

confidence: 90%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Mistranslating tRNA identifies a deleterious S213P mutation in theSaccharomyces cerevisiae eco1-1allele

Zhu

Berg

Yang

et al. 2020

Preprint

Self Cite

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“…This would provide evidence that native mistranslation provokes similar sensitivities and requires a response similar to the incorporation of a non-proteinogenic amino acid. Using a synthetic genetic array (Tong et al 2001), we measured the fitness of double mutants containing one of the 72 alleles identified above in combination with a mistranslating tRNA Ser variant with a UGG anticodon (Berg et al 2017(Berg et al , 2019. This variant mis-incorporates serine at proline codons at ~ 5% frequency and results in a 20% decrease in growth compared to a wildtype strain (Berg et al 2019).…”

Section: Similar Cellular Processes Are Required For Resistance To Otmentioning

confidence: 99%

Chemical-genetic interactions with the proline analog L-azetidine-2-carboxylic acid inSaccharomyces cerevisiae

Berg

Zhu

Isaacson

et al. 2020

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Non-proteinogenic amino acids, such as the proline analog L-azetidine-2-carboxylic acid (AZC), are detrimental to cells because they are mis-incorporated into proteins and lead to proteotoxic stress. Our goal was to identify genes that show chemical-genetic interactions with AZC in Saccharomyces cerevisiae and thus also potentially define the pathways cells use to cope with amino acid mis-incorporation. Screening the yeast deletion and temperature sensitive collections, we found 72 alleles with negative synthetic interactions with AZC treatment and 12 alleles that suppress AZC toxicity. Many of the genes with negative synthetic interactions are involved in protein quality control pathways through the proteasome. Genes involved in actin cytoskeleton organization and endocytosis also had negative synthetic interactions with AZC. Related to this, the number of actin patches per cell increases upon AZC treatment. Many of the same cellular processes were identified to have interactions with proteotoxic stress caused by two other amino acid analogs, canavanine and thialysine, or a mistranslating tRNA variant that mis-incorporates serine at proline codons. Alleles that suppressed AZC-induced toxicity functioned through the amino acid sensing TOR pathway or controlled amino acid permeases required for AZC uptake.

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Regulating Expression of Mistranslating tRNAs by Readthrough RNA Polymerase II Transcription

et al. 2021

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Transfer RNA (tRNA) variants that alter the genetic code increase protein diversity and have many applications in synthetic biology. Since the tRNA variants can cause a loss of proteostasis, regulating their expression is necessary to achieve high levels of novel protein. 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 tRNASer variant that misincorporates serine at proline codons in Saccharomyces cerevisiae allows mistranslation at a level not otherwise possible because of the toxicity of the unregulated tRNA. Using this inducible tRNA system, we explore the proteotoxic effects of mistranslation on yeast cells. High levels of mistranslation cause cells to arrest in the G1 phase. These cells are impermeable to propidium iodide, yet growth is not restored upon repressing tRNA expression. High levels of mistranslation increase cell size and alter cell morphology. This regulatable tRNA expression system can be applied to study how native tRNAs and tRNA variants affect the proteome and other biological processes. Variations of this inducible tRNA system should be applicable to other eukaryotic cell types.

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Modulating Mistranslation Potential of tRNASer inSaccharomyces cerevisiae

Abstract: Mistranslation, incorporating an amino acid not specified by the “standard” genetic code, has applications in research and synthetic biology. Since mistranslation is toxic, its level must be modulated. Using a serine tRNA with a proline anticodon, we identify...

Cited by 25 publications

References 90 publications

Mistranslating tRNA identifies a deleterious S213P mutation in theSaccharomyces cerevisiae eco1-1allele

Mistranslating tRNA identifies a deleterious S213P mutation in theSaccharomyces cerevisiae eco1-1allele

Chemical-genetic interactions with the proline analog L-azetidine-2-carboxylic acid inSaccharomyces cerevisiae

Regulating Expression of Mistranslating tRNAs by Readthrough RNA Polymerase II Transcription

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