thiouridine and 4-azidophenacyl-bromide

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.

Topics: Affinity Labels; Azides; Binding Sites; Diphosphonates; Escherichia coli; Guanosine Monophosphate; Guanosine Triphosphate; Kinetics; Magnesium; Mercaptoethanol; Peptide Elongation Factor Tu; Peptide Elongation Factors; Photolysis; Ribosomes; RNA, Transfer, Amino Acyl; Thiouridine

Phe-tRNA of Escherichia coli, specifically derivatized at the S4U8 position with the 9 A long p-azidophenacyl photoaffinity probe, can be crosslinked to 30 S ribosomal protein when the tRNA is placed at the ribosomal A site. This protein has now been identified by immunological methods. The protein-[3H]Phe-tRNA covalent complex, obtained by extraction with 6 M-urea, was reacted separately with each of the 21 purified antisera to 30 S ribosomal proteins. The double antibody technique was used. Anti-S19 was the only antiserum able to precipitate the radioactivity, and 66 to 81% of the added radioactivity could be precipitated. The same result was obtained with three different ribosome preparations, at low as well as high crosslinking yield, with dipeptidyl-tRNA in the A site as well as aminoacyl-tRNA, and when binding and crosslinking were performed at 20 mM-Mg2+ instead of at 5 mM. Therefore, when aminoacyl-tRNA or peptidyl-tRNA is in the ribosomal A site, position 8, which is always uridine or 4-thiouridine, must be within 9 A of protein S19.

Topics: Affinity Labels; Antibodies; Azides; Binding Sites; Centrifugation, Density Gradient; Escherichia coli; Peptide Elongation Factor Tu; Peptide Elongation Factors; Ribosomal Proteins; Ribosomes; RNA, Transfer, Amino Acyl; Thiouridine