Unique Residues Crucial for Optimal Editing in Yeast Cytoplasmic Leucyl-tRNA Synthetase Are Revealed by Using a Novel Knockout Yeast Strain

Peng Yao,Xiao-Long Zhou,Ran He,Mei-Qin Xue,Yong-Gang Zheng,Yue-Fei Wang,En-Duo Wang

doi:10.1074/jbc.m801181200

Abstract

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.

Highlights

It is presumed that the genetic code underwent an initial period during which codons did not specify an unalterable amino acid
We previously found that the human cytoplasmic Leucyl-tRNA synthetase (LeuRS) is quite active as a free enzyme, and its kinetic constants for ATP, leucine, and tRNALeu in the ATP-PPi exchange and tRNA leucylation reactions were determined [21]; the editing function of this enzyme has not yet been addressed
We define three types of residues based on conservation: 1) absolutely conserved residues through all species, e.g. Thr-319 and Asp-419; 2) conserved residues in the eukaryotic cytoplasmic LeuRSs from yeast to human, e.g. Thr347 and Thr-410; and 3) conserved residues of each kingdom, e.g. Lys-404, Ser-416, and Asp-418

Summary

Introduction

It is presumed that the genetic code underwent an initial period during which codons did not specify an unalterable amino acid. The molecular mechanisms of the editing function of prokaryotic LeuRS have been investigated in vitro (6 –11), and the crystallographic structure of prokaryotic or archaeal LeuRS complexed with the cognate tRNA has been reported [12, 13]. We previously found that the human cytoplasmic LeuRS (hcLeuRS) is quite active as a free enzyme, and its kinetic constants for ATP, leucine, and tRNALeu in the ATP-PPi exchange and tRNA leucylation reactions were determined [21]; the editing function of this enzyme has not yet been addressed. YcLeuRS performs the editing function through both pretransfer hydrolysis as a major pathway in vitro [24] and posttransfer editing in dependence on an essential conserved aspartic acid residue [9]. Our results suggest that several unique residues in ycLeuRS play important roles in optimal editing reaction to maintain translational fidelity in vivo. The in vivo data obtained by this method match up to those obtained in vitro, and the former seemed to be more sensitive to slight changes in the editing activity of ycLeuRS than the latter

Results

Discussion

Conclusion