Conditional accumulation of toxic tRNAs to cause amino acid misincorporation

Nucleic Acids Research
Stephanie M ZimmermanEric M Phizicky

Abstract

To develop a system for conditional amino acid misincorporation, we engineered tRNAs in the yeast Saccharomyces cerevisiae to be substrates of the rapid tRNA decay (RTD) pathway, such that they accumulate when RTD is turned off. We used this system to test the effects on growth of a library of tRNASer variants with all possible anticodons, and show that many are lethal when RTD is inhibited and the tRNA accumulates. Using mass spectrometry, we measured serine misincorporation in yeast containing each of six tRNA variants, and for five of them identified hundreds of peptides with serine substitutions at the targeted amino acid sites. Unexpectedly, we found that there is not a simple correlation between toxicity and the level of serine misincorporation; in particular, high levels of serine misincorporation can occur at cysteine residues without obvious growth defects. We also showed that toxic tRNAs can be used as a tool to identify sequence variants that reduce tRNA function. Finally, we generalized this method to another tRNA species, and generated conditionally toxic tRNATyr variants in a similar manner. This method should facilitate the study of tRNA biology and provide a tool to probe the effects of amino acid misincorporati...Continue Reading

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Citations

Jan 16, 2019·The Journal of Biological Chemistry·Jeremy T LantPatrick O'Donoghue
May 29, 2019·IUBMB Life·Rebecca E Steiner, Michael Ibba
Aug 14, 2019·RNA Biology·Matthew D BergChristopher J Brandl
Sep 6, 2019·Genetics·Matthew D BergChristopher J Brandl
Sep 10, 2020·RNA Biology·Matthew D Berg, Christopher J Brandl
Jun 2, 2020·Biochemistry and Cell Biology = Biochimie Et Biologie Cellulaire·Yanrui ZhuChristopher J Brandl
Nov 28, 2018·International Journal of Molecular Sciences·Marisa PereiraAna Raquel Soares
Oct 22, 2020·G3 : Genes - Genomes - Genetics·Matthew D BergChristopher J Brandl
Nov 1, 2019·Genetics·Matthew D BergChristopher J Brandl
Nov 3, 2021·ACS Synthetic Biology·Matthew D BergChristopher J Brandl

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Methods Mentioned

BETA
PCR
Protein Assay
acetylation
Assay

Software Mentioned

Bowtie2
Percolator
Comet

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