Photochemical cross-linking of unmodified acetylvalyl-tRNA to 16S RNA at the ribosomal P site.
Acetylvalyl-, acetylphenylalanyl-, and formylmethionyl-tRNA which were derivatized at their 4-thiouridine residues with the photoaffinity label, p-azidophenacyl bromide, were nonenzymatically bound to salt-washed ribosomes. More than 90% of the binding was to the P site as judged by reactivity with puromycin. Subsequent irradiation (greater than 310 nm) of the tRNA-ribosome complexes resulted in the covalent linking of only the acetylvalyl-tRNA to the 30S subunit. Attachment was solely to the 16S RNA with an efficiency of cross-linking of 13--15%. Covalent linking was 90% inhibited by prior treatment with puromycin, showing that the covalent linking reaction had taken place at the P site. Cross-linking required irradiation and mRNA but was not dependent on the presence of the photoaffinity probe in the tRNA. tRNAs whose 4-thiouridine had been modified with unreactive analogues of p-azidophenacyl bromide or unmodified acetylvalyl-tRNA exhibited the same cross-linking behavior as photoaffinity probe-modified acetylvalyl-tRNA. Furthermore, even acetylvalyl-tRNA whose 4-thiouridine had been removed by treatment with H2O2 was quantitatively as active as unmodified tRNA. These results provide the first demonstration of direct photochemical cross-linking of tRNA to ribosomes.
D000345:Affinity Labels; D001665:Binding Sites; D004926:Escherichia coli / Q000378:metabolism*; D007700:Kinetics; D008970:Molecular Weight; D010649:Phenylalanine / Q000031:analogs & derivatives; D010777:Photochemistry; D011691:Puromycin; D012335:RNA, Ribosomal / Q000378:metabolism; D012346:RNA, Transfer, Amino Acyl / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism*; D014633:Valine / Q000031:analogs & derivatives
D000345:Affinity Labels; D012335:RNA, Ribosomal; D012346:RNA, Transfer, Amino Acyl; D010649:Phenylalanine; D011691:Puromycin; D014633:Valine