PMID:11434781
| Citation |
Tardif, KD, Liu, M, Vitseva, O, Hou, YM and Horowitz, J (2001) Misacylation and editing by Escherichia coli valyl-tRNA synthetase: evidence for two tRNA binding sites. Biochemistry 40:8118-25 |
|---|---|
| Abstract |
Valyl-tRNA synthetase (ValRS) has difficulty discriminating between its cognate amino acid, valine, and structurally similar amino acids. To minimize translational errors, the enzyme catalyzes a tRNA-dependent editing reaction that prevents accumulation of misacylated tRNA(Val). Editing occurs with threonine, alanine, serine, and cysteine, as well as with several nonprotein amino acids. The 3'-end of tRNA plays a vital role in promoting the tRNA-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, particularly 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'-pyrimidines 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 does 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) are mixed noncompetitive inhibitors of the aminoacylation reaction, suggesting that ValRS, a monomeric enzyme, may bind more than one tRNA(Val) molecule. Gel-mobility-shift experiments to characterize the interaction of tRNA(Val) with the enzyme provide evidence for two tRNA binding sites on ValRS. |
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| Keywords |
3' Untranslated Regions/metabolism; Acylation; Binding Sites; Escherichia coli/enzymology; Escherichia coli/genetics; Hydrolysis; Isoleucine/metabolism; RNA Editing; RNA, Bacterial/metabolism; RNA, Transfer, Amino Acyl/metabolism; RNA, Transfer, Val/metabolism; Transfer RNA Aminoacylation; Valine-tRNA Ligase/antagonists & inhibitors; Valine-tRNA Ligase/metabolism |
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