Misacylation and editing by Escherichia coli valyl-tRNA synthetase: evidence for two tRNA binding sites.
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.
Tardif
K D
KD
Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University, Ames, Iowa 50011, USA.
Liu
M
M
Vitseva
O
O
Hou
Y M
YM
Horowitz
J
J
D020413:3' Untranslated Regions / Q000378:metabolism; D000215:Acylation; D001665:Binding Sites; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D006868:Hydrolysis; D007532:Isoleucine / Q000378:metabolism; D017393:RNA Editing; D012329:RNA, Bacterial / Q000378:metabolism; D012346:RNA, Transfer, Amino Acyl / Q000378:metabolism; D012366:RNA, Transfer, Val / Q000378:metabolism*; D046249:Transfer RNA Aminoacylation; D014637:Valine-tRNA Ligase / Q000037:antagonists & inhibitors / Q000378:metabolism*
D020413:3' Untranslated Regions; D012329:RNA, Bacterial; D012346:RNA, Transfer, Amino Acyl; D012366:RNA, Transfer, Val; D007532:Isoleucine; D014637:Valine-tRNA Ligase