The prime function of leucyl-tRNA synthetase proofreading is prevention of the non-canonical mistranslation in Escherichia coli

Aminoacyl-tRNA synthetases (aaRSs) are enzymes that covalently pair amino acids with cognate tRNAs in a two-step reaction. Amino acid is firstly activated through formation of aminoacyl-adenylate, and in the second step, aminoacyl-moiety is transferred to cognate tRNA. Coupling of cognate aa-tRNA pairs is crucial for accurate protein biosynthesis. Selection of the cognate tRNA generally does not pose a problem for aaRSs, as they share a large interacting surface with the tRNA molecules. However, some structurally similar amino acids are difficult to discriminate in the synthetic reaction alone. AaRSs have developed complex proofreading mechanisms for correction of errors made in amino acid selection that may occur before or after transfer of amino acid to tRNA (hydrolysis of aminoacyl-adenylate or misacylated tRNA, respectively). Leucyl-tRNA synthetases (LeuRS) covalently couple tRNALeu with leucine, and thereby provide the pool of Leu-tRNALeu for ribosomal protein synthesis. LeuRS may also activate and transfer to tRNALeu structurally and chemically similar norvaline, a non–canonical amino acid that accumulates in Escherichia coli under micro-aerobic conditions. However, incorporation of norvaline into proteins is prevented by efficient intrinsic LeuRS hydrolytic activity toward norvalyl-tRNALeu within a dedicated post-transfer editing domain. In spite of the prevailing opinion that noncognate isoleucine mimics leucine well in the LeuRS synthetic reactions and thus requires editing to prevent errors in leucyl-tRNALeu synthesis, we now demonstrate that isoleucine is discriminated with high specificity within the synthetic site. Thermodynamic, structural and kinetic approaches establish that both very weak ground state binding and the decreased rate of the chemical step contribute to isoleucine discrimination. These results were complemented by in vivo experiments, showing that E. coli strain relying on the LeuRS with defunct post-transfer editing domain exhibits normal growth in the presence of high isoleucine concentration, but displays growth defects under micro-aerobic conditions where norvaline accumulates. Our results reveal that LeuRS mediated translational quality control represents the essential part of the major E. coli adaptive response necessary for survival in environments with low oxygen levels.