Covalent crosslinking of Escherichia coli phenylalanyl-tRNA and valyl-tRNA to the ribosomal A site via photoaffinity probes attached to the 4-thiouridine residue.
tRNAPhe and tRNAVal of Escherichia coli were derivatized at the S4U8 position with p-azidophenacyl and p-azidophenacylacetate photoaffinity probes. The modified tRNAs could still function efficiently in all of the partial reactions of protein synthesis except for an approximately sevenfold decrease in the rate of translocation. Irradiation (310 to 340 nm) of probe-modified Phe-tRNA or Val-tRNA placed in the ribosomal A site led to crosslinking that was totally dependent on irradiation, the presence of the azido group on the probe, mRNA, and elongation factor Tu (EFTu). Prephotolysis of the modified tRNA abolished crosslinking, but prephotolysis of the ribosomes and factors had little effect. Crosslinking was efficiently quenched by mercaptoethanol or dithiothreitol, demonstrating accessibility of the probe to solvent. Use of GDPCP in place of GTP also blocked crosslinking, probably because of the retention of EFTu on the ribosome. Crosslinking with the p-azidophenacyl acetate (12 A) probe was only half as efficient as with the p-azidophenacyl (9 A) probe, and this ratio was not changed by varying Mg2+ from 5 to 15 mM. The crosslink was from a functional A site, since AcPhePhe-tRNA at the A site could be crosslinked, and it was A site-specific, because neither translocation nor direct non-enzymatic P site binding yielded any significant covalent product. The crosslink was to ribosomal protein(s) of the 30 S subunit. No other ribosomal component was crosslinked. Identification of the protein crosslinked is described in the accompanying paper.
D000345:Affinity Labels / Q000494:pharmacology; D001386:Azides / Q000494:pharmacology; D001665:Binding Sites / Q000187:drug effects; D004164:Diphosphonates / Q000494:pharmacology; D004926:Escherichia coli; D006157:Guanosine Monophosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D006160:Guanosine Triphosphate / Q000494:pharmacology / Q000031:analogs & derivatives; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008623:Mercaptoethanol / Q000494:pharmacology; D010444:Peptide Elongation Factor Tu; D010445:Peptide Elongation Factors / Q000494:pharmacology; D010782:Photolysis; D012346:RNA, Transfer, Amino Acyl / Q000378:metabolism*; D012270:Ribosomes / Q000378:metabolism*; D013891:Thiouridine / Q000378:metabolism*
D000345:Affinity Labels; D001386:Azides; D004164:Diphosphonates; D010445:Peptide Elongation Factors; D012346:RNA, Transfer, Amino Acyl; D013891:Thiouridine; C004805:5'-guanylylmethylenebisphosphonate; D008623:Mercaptoethanol; D006157:Guanosine Monophosphate; D006160:Guanosine Triphosphate; D010444:Peptide Elongation Factor Tu; D008274:Magnesium; C011171:4-azidophenacyl bromide