In vivo identification of essential nucleotides in tRNA(Leu) to its functions by using a constructed yeast tRNA(Leu) knockout strain

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 tRNA(Leu) 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 tRNA(Leu) knockout strains carrying deletions of the genes for tRNA(Leu)(GAG) and tRNA(Leu)(UAG). Disrupting the single gene encoding tRNA(Leu)(GAG) had no phenotypic consequence when compared to the wild-type strain. While disrupting the three genes for tRNA(Leu)(UAG) had a lethal effect on the yeast strain, indicating that tRNA(Leu)(UAG) decoding capacity could not be compensated by another tRNA(Leu) isoacceptor. Using the triple tRNA knockout strain and a randomly mutated library of tRNA(Leu)(UAG), a selection to identify critical tRNA(Leu) 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.