Novel Function of Yeast tRNALeu Revealed by Yeast Knockout Strains
Source:
Time: 2012-08-30
On August 23rd, 2012 Nucleic Acids Research online published the work entitled “In vivo identification of essential nucleotides in tRNALeu to its functions by using a constructed yeast tRNALeu knockout strain” from Prof. WANG Enduo’s group in Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences..
Aminoacyl-tRNA synthetases (aaRSs) catalyze aminoacylation of their cognate tRNAs with amino acids to form correct aminoacyl-tRNAs, which are the substrates of protein biosynthesis in ribosome. The fidelity of the catalytic process requires accurate interaction between aaRS and its cognate tRNA. Crucial bases in tRNALeu to aminoacylation or editing functions of leucyl-tRNA synthetase (LeuRS) have been extensively studied mainly by in vitro methods, including T7 RNA transcription and site directed mutagenesis. However, in vitro transcribed tRNAs may have low activity due to the absence of critical base modifications; and to obtain the mutants of tRNA by point-by-point site directed mutagenesis method consumes more times and labors. Transcripts from S. cerevisiae tRNALeu usually are very poor substrates for LeuRS, thus few papers about the interaction between ycLeuRS and tRNALeu were reported.
HUANG Qian and his colleagues guided by Professor WANG Enduo constructed two yeast knockout strains- tl(gag)-Δ1 and tl(uag)-Δ1-3 in vivo, deleted tRNALeu(GAG) and tRNALeu(UAG), respectively. They found that tRNALeu(GAG) is an redundant tRNA, tRNALeu(UAG) could translate the 4 CUN codons, being an essential tRNA in vivo. This "superwobbling" phenomenon was first found in yeast. They constructed a random mutation library and performed a genetic selection using tl(uag)-Δ1-3. Mutations inducing in vivo decreases of tRNA levels or aminoacylation or editing ability by LeuRS were identified. Additionally, the tRNALeu(UAG) mutants with the modified bases were obtained from yeast tl(uag)-Δ1-3 transformants, and in vitro kinetic and editing experiments were performed. The in vivo and in vitro data fit well. Overall, the results showed that the triple tRNALeu knockout strain is a suitable tool for in vivo studies and identification of essential nucleotides of the tRNALeu.
This study was funded by grants from the National Basic Research Program of China and the National Natural Science Foundation of China.
AUTHOR CONTACT:
WANG Enduo
Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, Shanghai, China
Phone: 86-21-54921241; E-mail: edwang@sibs.ac.cn
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