PROOFREADING IN TRANS BY AN AMINOACYL-TRNA SYNTHETASE - A MODEL FOR SINGLE SITE EDITING BY ISOLEUCYL-TRNA SYNTHETASE

摘要

Editing of errors in amino acid selection by an aminoacyl-tRNA synthetase prevents attachment of incorrect amino acids to tRNA, thereby greatly enhancing accuracy of translation of the genetic code, Editing of the non-protein amino acid homocysteine, a frequent type of an error-correcting process, involves reaction of the side chain sulfhydryl group of homocysteine with its activated carboxyl group forming a cyclic thioester, homocysteine thiolactone. Here, it is shown that isoleucyl-tRNA synthetase (IleRS), which occasionally misactivates homocysteine in vitro and in vivo, catalyzes reactions of activated isoleucine with organic thiols (analogues of the side chain of homocysteine), That these enzymatic reactions occur between Ile-tRNA(Ile) or Ile-AMP (bound in the synthetic sub-site) and a thiol (an analogue of the side chain of homocysteine, bound in the editing sub-site), indicates that the two sub-sites are physically close on the surface of IleRS, forming a single synthetic/editing active site of the enzyme, Although IleRS . Val-AMP undergoes thiolysis as efficiently as do IleRS . Ile-AMP and IleRS . Ile-tRNA(Ile), IleRS . Val-tRNA(Ile) does not react with thiols, These and other data suggest that the mischarged valine residue in IleRS . Val-tRNA(Ile) is, most likely, positioned off the enzyme.