Preparation of Misacylated Aminoacyl-tRNAPhe's Useful as Probes of the Ribosomal Acceptor Site

James R. Roesser, Cheng Xu, Robert C. Payne, Christopher K. Surratt, Sidney M. Hecht

Research output: Contribution to journalArticle

66 Scopus citations

Abstract

Several pyroglutamylaminoacyl-tRNA's were prepared by T4 RNA ligase mediated condensation of synthetic pyroglutamylaminoacyl-pCpA's with tRNA's from which the last two nucleotides at the 3'-end had been removed. The derived pyroglutamylaminoacyl-tRNA's were incubated in the presence of calf liver pyroglutamate aminopeptidase, which effected their conversion to free aminoacyl-tRNA's. The lack of contaminating esterase activities in the pyroglutamate aminopeptidase was verified by direct assay for the presence of the aminoacyl moieties in the formed aminoacyl-tRNA's and by the use of the deblocked aminoacyl-tRNA's as acceptors in the peptidyltransferase reaction using an Escherichia coli ribosomal system. These findings provide the wherewithal for a detailed investigation of the substrate specificity of the peptidyltransferase center and for the elaboration of polypeptides containing modified amino acids at predetermined sites.

Original languageEnglish (US)
Pages (from-to)5185-5195
Number of pages11
JournalBiochemistry
Volume28
Issue number12
DOIs
StatePublished - 1989

ASJC Scopus subject areas

  • Biochemistry

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