Photo-Affinity Labeling of tRNA Binding Sites in Macromolecules. I. Linking of the Phenacyl- p -azide of 4-Thiouridine in ( Escherichia coli ) Valyl-tRNA to 16S RNA at the Ribosomal P Site

The phenacyl-p-azide of 4-thiouridine in (E. coli) tRNA(1) (Val) was prepared for use as a photo-affinity probe of tRNA binding sites on ribosomes. The derivatized tRNA was 90-100% as active as control tRNA for aminoacylation, nonenzymatic binding to the ribosomal P site, elongation factor Tu(EFTu)-dependent binding to the A site, EFTu-GTP-aa-tRNA ternary complex formation, and transfer of valine into polypeptide. Irradiation of p-azidophenacyl-[(3)H]valyl-tRNA bound noncovalently to the ribosomal P site resulted in covalent attachment of 15-20% of the noncovalently bound tRNA to the ribosomes. The linking occurred exclusively to the 16S RNA of the 30S ribosomal subunit, thus suggesting that the region of the ribosome within 9 A of the 4-thiouridine of tRNA, when it is bound in the P site, is solely 16S RNA.