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

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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 contamining 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.