Unique residues crucial for optimal editing in yeast cytoplasmic Leucyl-tRNA synthetase are revealed by using a novel knockout yeast strain.
Leucyl-tRNA synthetase (LeuRS) contains an editing domain that discriminates leucine from noncognate amino acids to ensure translational fidelity. In this study, a knock-out strain for Saccharomyces cerevisiae LeuRS was constructed to analyze in vivo the tRNA aminoacylation properties of S. cerevisiae and human cytoplasmic LeuRSs. The activities of several editing-defective mutants of ycLeuRS were determined in vitro and compared with those obtained in vivo in a complementation assay performed in the knock-out strain. The editing activities of these mutants were analyzed in the presence of either norvaline, a leucine analogue, or AN2690, a specific inhibitor that targets the editing active site. In general, the in vivo data are consistent with those obtained in vitro. Our results show that ycLeuRS post-transfer editing plays a crucial role in the establishment of the aminoacylation fidelity. When impaired, the viability of cells bearing editing-defective mutants is drastically decreased in the presence of noncognate amino acid. This study also emphasizes the crucial function of some semi-conserved residues around the editing site in modulating the editing efficiency. The assay system can be used to test the effect of compounds that potentially target the aminoacylation or editing active site of fungal LeuRS.
State Key Laboratory of Molecular Biology, Graduate School of the Chinese Academy of Sciences, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, The Chinese Academy of Sciences, Shanghai, China.
Peng
P
Yao
Xiao-Long
XL
Zhou
Ran
R
He
Mei-Qin
MQ
Xue
Yong-Gang
YG
Zheng
Yue-Fei
YF
Wang
En-Duo
ED
Wang
D017931:DNA Primers; D004271:DNA, Fungal / Q000235:genetics; D004789:Enzyme Activation; D007700:Kinetics; D007935:Leucine-tRNA Ligase / Q000172:deficiency / Q000235:genetics* / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D017393:RNA Editing; D012331:RNA, Fungal / Q000235:genetics; D011994:Recombinant Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism
D017931:DNA Primers; D004271:DNA, Fungal; D012331:RNA, Fungal; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D007935:Leucine-tRNA Ligase