Dataset Information

Conditional accumulation of toxic tRNAs to cause amino acid misincorporation.

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.

SUBMITTER: Zimmerman SM

PROVIDER: S-EPMC6125640 | biostudies-literature | 2018 Sep

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Conditional accumulation of toxic tRNAs to cause amino acid misincorporation.

Zimmerman Stephanie M SM Kon Yoshiko Y Hauke Alayna C AC Ruiz Bianca Y BY Fields Stanley S Phizicky Eric M EM

Nucleic acids research 20180901 15

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 ...[more]

PMID: 30007351

Similar Datasets

Project description:Codon usage frequency influences protein structure and function. The frequency with which codons are used potentially impacts primary, secondary and tertiary protein structure. Poor expression, loss of function, insolubility, or truncation can result from species-specific differences in codon usage. "Codon harmonization" more closely aligns native codon usage frequencies with those of the expression host particularly within putative inter-domain segments where slower rates of translation may play a role in protein folding. Heterologous expression of Plasmodium falciparum genes in Escherichia coli has been a challenge due to their AT-rich codon bias and the highly repetitive DNA sequences. Here, codon harmonization was applied to the malarial antigen, CelTOS (Cell-traversal protein for ookinetes and sporozoites). CelTOS is a highly conserved P. falciparum protein involved in cellular traversal through mosquito and vertebrate host cells. It reversibly refolds after thermal denaturation making it a desirable malarial vaccine candidate. Protein expressed in E. coli from a codon harmonized sequence of P. falciparum CelTOS (CH-PfCelTOS) was compared with protein expressed from the native codon sequence (N-PfCelTOS) to assess the impact of codon usage on protein expression levels, solubility, yield, stability, structural integrity, recognition with CelTOS-specific mAbs and immunogenicity in mice. While the translated proteins were expected to be identical, the translated products produced from the codon-harmonized sequence differed in helical content and showed a smaller distribution of polypeptides in mass spectra indicating lower heterogeneity of the codon harmonized version and fewer amino acid misincorporations. Substitutions of hydrophobic-to-hydrophobic amino acid were observed more commonly than any other. CH-PfCelTOS induced significantly higher antibody levels compared with N-PfCelTOS; however, no significant differences in either IFN-? or IL-4 cellular responses were detected between the two antigens.

Dataset Information

Conditional accumulation of toxic tRNAs to cause amino acid misincorporation.

Publications

Conditional accumulation of toxic tRNAs to cause amino acid misincorporation.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets