| Aminoacyl-tRNA synthetases are responsible for the esterification of cognate amino acids to their specific tRNAs. To maintain protein synthesis fidelity, these enzymes must distinguish between closely related substrates. Leucyl-tRNA synthetase (LeuRS) activates a wide array of amino acids, but contains a hydrolytic editing domain called CP1 to enhance fidelity. We screened all twenty standard amino acids and several other cellular metabolic intermediates to determine which amino acids actually threaten LeuRS fidelity in vivo. We used a temperature-sensitive E. coli strain and introduced editing-defective LeuRS mutants. Our results show that cellular viability is dependent on the LeuRS editing activity to suppress toxicity by isoleucine, valine, methionine, norvaline, norleucine, homocysteine and homoserine.;Different editing-defective mutants confer varied thresholds of fidelity in vitro. We hypothesized that these editing defects might also have varied effects in vivo. We transformed a temperature-sensitive E. coli strain with LeuRS genes that encoded different editing mutants. These included single mutations of amino acids throughout the editing active site, as well as combination of mutations. Significantly, E. coli cells harboring double and triple editing-defective LeuRS mutants had a greater effect on their viability compared to the single mutants emphasizing the physiological importance of these residues.;We tested several mutations in yeast mitochondrial LeuRS that altered the post-transfer editing function of LeuRSs from other origins. Our results show that yeast mitochondrial LeuRS has maintained a competent editing active site for post-transfer editing of mischarged tRNA similar to other LeuRSs. However, unlike other origins, when LeuRS amino acid editing-defective mutants were introduced in vivo, cell viability was largely unaffected. In contrast, these editing-defective mutations limited viability of E. coli cells. We propose that the yeast mitochondria have evolved to tolerate lower levels of fidelity in protein synthesis. |
