Conditional accumulation of toxic tRNAs to cause amino acid misincorporation.
Nucleic Acids Res
; 46(15): 7831-7843, 2018 09 06.
Article
en En
| MEDLINE
| ID: mdl-30007351
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 misincorporation on cellular physiology.
Texto completo:
1
Colección:
01-internacional
Banco de datos:
MEDLINE
Asunto principal:
Saccharomyces cerevisiae
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Biosíntesis de Proteínas
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ARN de Transferencia de Serina
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ARN de Transferencia de Tirosina
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Sustitución de Aminoácidos
Idioma:
En
Revista:
Nucleic Acids Res
Año:
2018
Tipo del documento:
Article
País de afiliación:
Estados Unidos