Abstract
The rules of the genetic code are established in reactions that aminoacylate tRNAs with specific amino acids. Ambiguity in the code is prevented by editing activities whereby incorrect aminoacylations are cleared by specialized hydrolytic reactions of aminoacyl tRNA synthetases. Whereas editing reactions have long been known, their significance for cell viability is still poorly understood. Here we investigated in vitro and in vivo four different mutations in the center for editing that diminish the proofreading activity of valyl-tRNA synthetase (ValRS). The four mutant enzymes were shown to differ quantitatively in the severity of the defect in their ability to clear mischarged tRNA in vitro. Strikingly, in the presence of excess concentrations of alpha-aminobutyrate, one of the amino acids that is misactivated by ValRS, growth of bacterial strains bearing these mutant alleles is arrested. The concentration of misactivated amino acid required for growth arrest correlates inversely in a rank order with the degree of the editing defect seen in vitro. Thus, cell viability depends directly on the suppression of genetic code ambiguity by these specific editing reactions and is finely tuned to any perturbation of these reactions.
Highlights
The rules of the genetic code are established in reactions that aminoacylate tRNAs with specific amino acids
We investigated in vitro and in vivo four different mutations in the center for editing that diminish the proofreading activity of valyl-tRNA synthetase (ValRS)
Aminoacyl-tRNA synthetases (AARSs)1 catalyze the attachment of amino acids to their cognate tRNAs to establish the genetic code
Summary
The rules of the genetic code are established in reactions that aminoacylate tRNAs with specific amino acids. We investigated in vitro and in vivo four different mutations in the center for editing that diminish the proofreading activity of valyl-tRNA synthetase (ValRS). ValRS catalyzes specific aminoacylation of tRNAVal in a reaction initiated by the binding of valine at the active site where it is condensed with ATP to form valyl adenylate (Val-AMP). Given that the editing domain is conserved through evolution, including the most ancient organisms in the bacterial and archaebacterial kingdoms [26], we imagined that cell growth would be sensitive to defects in the editing activity These mutant enzymes afforded an opportunity to assess the relationship between the degree of the editing defect observed in vitro and the growth phenotype or toxicity seen in vivo. The mutant enzymes provided further opportunity to evaluate the chemical role that specific conserved residues might have in the editing activity
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