Misacylation and editing by Escherichia coli Valyl-tRNA synthetase: Evidence for two tRNA binding sites

Valyl-tRNA synthetase (ValRS) has difficulty discriminating between its cog nate amino acid, valine, and structurally similar amino acids. To minimize translational errors. the enzyme catalyzes a tRNA-dependent editing reactio n that prevents accumulation of misacylated tRNA(Val). Editing occurs with threonine, alanine, serine, and cysteine, as well as with several nonprotei n amino acids. The 3 ' -end of tRNA plays a vital role in promoting the tRN A-dependent editing reaction. Valine tRNA having the universally conserved 3 ' -terminal adenosine replaced by any other nucleoside does not stimulate the editing activity of ValRS, As a result 3 ' -end tRNA(Val) mutants, par ticularly those with 3 ' -terminal pyrimidines, are stably misacylated with threonine, alanine, serine, and cysteine. Valyl-tRNA synthetase is unable to hydrolytically deacylate misacylated tRNA(Val) terminating in 3 ' -pyrim idines but does deacylate mischarged tRNA(Val) terminating in adenosine or guanosine. Evidently, a purine at position 76 of tRNA(Val) is essential for translational editing by ValRS. We also observe misacylation of wild-type and 3 ' -end mutants of tRNA(Val) with isoleucine. Valyl-tRNA synthetase do es not edit wild-type tRNA(Val)(A76) mischarged with isoleucine, presumably because isoleucine is only poorly accommodated at the editing site of the enzyme. Misacylated mutant tRNAs as well as 3 ' -end-truncated tRNA(Val) ar e mixed noncompetitive inhibitors of the aminoacylation reaction, suggestin g that ValRS, a monomeric enzyme, may bind more than one tRNA(Val) molecule . Gel-mobility-shift experiments to characterize the interaction of tRNA(Va l) with the enzyme provide evidence for two tRNA binding sites on ValRS.