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Nucleic Acids Res. 2012 Nov 1;40(20):10463-77. doi: 10.1093/nar/gks783. Epub 2012 Aug 23.

In vivo identification of essential nucleotides in tRNALeu to its functions by using a constructed yeast tRNALeu knockout strain.

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  • 1Center for RNA research, State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, The Chinese Academy of Sciences, 320 Yue Yang Road, Shanghai 200031, China.

Abstract

The fidelity of protein biosynthesis requires the aminoacylation of tRNA with its cognate amino acid catalyzed by aminoacyl-tRNA synthetase with high levels of accuracy and efficiency. Crucial bases in tRNALeu to aminoacylation or editing functions of leucyl-tRNA synthetase have been extensively studied mainly by in vitro methods. In the present study, we constructed two Saccharomyces cerevisiae tRNALeu knockout strains carrying deletions of the genes for tRNALeu(GAG) and tRNALeu(UAG). Disrupting the single gene encoding tRNALeu(GAG) had no phenotypic consequence when compared to the wild-type strain. While disrupting the three genes for tRNALeu(UAG) had a lethal effect on the yeast strain, indicating that tRNALeu(UAG) decoding capacity could not be compensated by another tRNALeu isoacceptor. Using the triple tRNA knockout strain and a randomly mutated library of tRNALeu(UAG), a selection to identify critical tRNALeu elements was performed. In this way, mutations inducing in vivo decreases of tRNA levels or aminoacylation or editing ability by leucyl-tRNA synthetase were identified. Overall, the data showed that the triple tRNA knockout strain is a suitable tool for in vivo studies and identification of essential nucleotides of the tRNA.

PMID:
22917587
[PubMed - indexed for MEDLINE]
PMCID:
PMC3488233
Free PMC Article

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