Photoaffinity labeling of the ribosomal peptidyl transferase site with synthetic puromycin analogues.
A photoaffinity labeling puromycin analogue, Nepsilon-(2-nitro-4-azidophenyl)-L-lysinyl puromycin aminonucleoside (NAP-Lys-Pan), was synthesized and used for investigation of the peptidyl transferase center of 70S riobsomes. Visible light irradiation of NAP-Lys-Pan led to covalent linkage of the analogue with Escherichia coli ribosomes. In a subsequent step, poly(uridylic acid) was employed to direct Ac[14C]Phe-tRNA to the P sites of the photolabeled ribosomes. Transpeptidation of Ac[14C]phenylalanine to the bound NAP-Lys-Pan resulted in selective incorporation of radioactive label into the peptidyl transferase A site. Dissociation of the ribosomes into subunits, and digestion of the RNA components, indicated that the radioactive label was incorporated into a protein fraction of the 50S subunit.
D000217:Acyltransferases / Q000378:metabolism*; D000345:Affinity Labels; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D010458:Peptidyl Transferases / Q000378:metabolism*; D010782:Photolysis; D011691:Puromycin / Q000031:analogs & derivatives*; D012270:Ribosomes / Q000201:enzymology*
D000345:Affinity Labels; D011691:Puromycin; D000217:Acyltransferases; D010458:Peptidyl Transferases