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Detecting mistranslation in yeast expressing tRNA variants

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 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.

INSTRUMENT(S): Orbitrap Exploris 480

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

SUBMITTER: Matthew Berg

LAB HEAD: Dr. Judit Villen

PROVIDER: PXD025934 | Pride | 2021-05-17

REPOSITORIES: Pride

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			Action	DRS
	ArgSer10_CY8614.raw	Raw
	ArgSer11_CY8614.raw	Raw
	ArgSer12_CY8614.raw	Raw
	ArgSer25_CY8614.raw	Raw
	ArgSer27_CY8614.raw	Raw

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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.

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