The Bacterial YbaK Protein Is a Cys-tRNAPro andCys-tRNACysDeacylase

Benfang Ruan,Dieter Söll

doi:10.1074/jbc.m502174200

Abstract

Bacterial prolyl-tRNA synthetases and some smaller paralogs, YbaK and ProX, can hydrolyze misacylated Cys-tRNA Pro or Ala-tRNA Pro. To assess the significance of this quality control editing reaction in vivo, we tested Escherichia coli ybaK for its ability to suppress the E. coli thymidylate synthase thyA:146CCA missense mutant strain, which requires Cys-tRNA(Pro) for growth in the absence of thymine. Missense suppression was observed in a ybaK deletion background, suggesting that YbaK functions as a Cys-tRNA Pro deacylase in vivo. In vitro studies with the full set of 20 E. coli aminoacyl-tRNAs revealed that the Haemophilus influenzae and E. coli YbaK proteins are moderately general aminoacyl-tRNA deacylases that preferentially hydrolyze Cys-tRNA Pro and Cys-tRNA Cys and are also weak deacylases that cleave Gly-tRNA, Ala-tRNA, Ser-tRNA, Pro-tRNA, and Met-tRNA. The ProX protein acted as an aminoacyl-tRNA deacylase that cleaves preferentially Ala-tRNA and Gly-tRNA. The potential of H. influenzae YbaK to hydrolyze in vivo correctly charged Cys-tRNA Cys was tested in E. coli strain X2913 (ybaK+). Overexpression of H. influenzae ybaK decreased the in vivo ratio of Cys-tRNA Cys to tRNA Cys from 65 to 35% and reduced the growth rate of strain X2913 by 30% in LB medium. These data suggest that YbaK-mediated hydrolysis of aminoacyl-tRNA can influence cell growth.

Highlights

A high level of aminoacyl-tRNA1 is the basis for normal protein synthesis [1]. aa-tRNA formation is catalyzed by aminoacyl-tRNA synthetases that selectively attach one of the 20 canonical amino acids to the cognate tRNA species
The presence of additional E. coli ProRS makes strain X2914/thyA:146CCA/ pACYCtaq-ECproS grow slower than strain X2914/thyA: 146CCA; presumably, the higher amount of Cys-tRNAPro in the former strain is deleterious to the cell
General Discussion—The intracellular levels of aa-tRNAs are important. They affect the accuracy of translation [27], the rate of protein synthesis [28], and some reactions in intermediary metabolism (29 –31)

Summary

Introduction

A high level of aminoacyl-tRNA (aa-tRNA)1 is the basis for normal protein synthesis [1]. aa-tRNA formation is catalyzed by aminoacyl-tRNA synthetases (aaRSs) that selectively attach one of the 20 canonical amino acids to the cognate tRNA species. Bacterial prolyl-tRNA synthetases and some smaller paralogs, YbaK and ProX, can hydrolyze misacylated Cys-tRNAPro or Ala-tRNAPro. To assess the significance of this quality control editing reaction in vivo, we tested Escherichia coli ybaK for its ability to suppress the E. coli thymidylate synthase thyA:146CCA missense mutant strain, which requires Cys-tRNAPro for growth in the absence of thymine.

Results

Conclusion