Preparation of misacylated aminoacyl-tRNAPhe's useful as probes of the ribosomal acceptor site

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.