Preparation of misacylated aminoacyl-tRNA(Phe)'s useful as probes of the ribosomal acceptor site.
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.
Roesser
J R
JR
Department of Chemistry, University of Virginia, Charlottesville 22901.
Xu
C
C
Payne
R C
RC
Surratt
C K
CK
Hecht
S M
SM
D000215:Acylation; D001665:Binding Sites; D004926:Escherichia coli / Q000378:metabolism; D006868:Hydrolysis; D010452:Peptide Biosynthesis; D011751:Pyroglutamyl-Peptidase I / Q000378:metabolism; D011130:RNA Ligase (ATP) / Q000378:metabolism; D015347:RNA Probes; D012346:RNA, Transfer, Amino Acyl / Q000096:biosynthesis*; D012270:Ribosomes / Q000378:metabolism; D013604:T-Phages / Q000201:enzymology
D015347:RNA Probes; D012346:RNA, Transfer, Amino Acyl; D011751:Pyroglutamyl-Peptidase I; D011130:RNA Ligase (ATP)