Escherichia coli Phe-tRNA, modified with the photoaffinity reagent 6-(2-nitro-4-azidophenylamino)caproate on the 3-(3-amino-3-carboxypropyl)urindine residue, was crosslinked to E. coli EFTu upon irradiation of 0°C with visible light at wavelengths greater than 400nm. Crosslinking was dependent on irradiation, the photoaffinity probe, and was blocked by pre-photolysis. I mm-dithiothreitol completely quenched crosslinking. Binding of the tRNA to EFTu was a prerequisite for crosslinking, because neither EFTu·GDP nor AcPhe-tRNA could substitute; EFTu·GDPCP, however, was almost as active as EFTu·GTP. Crosslinking was complete in less than five minutes and was stable to at least 20 minutes of irradiation with a single 650 W tungsten lamp 4 cm away. The crosslinking yield ranged from 15% to 25%. The crosslinked complex possessed several remarkable properties. At 0·5 mm-Mg2+, the complex protected the AA-tRNA link to chemical hydrolysis, stabilized the bound GTP to dissociation or exchange, and was not adsorbed to cellulose nitrate filters. The purified crosslinked complex could be bound to ribosomes with concomitant hydrolysis of GTP. Extensive peptide bond formation with AcPhe-tRNA in the P site occurred despite the presence of the crosslinked EFTu. We conclude that hydrolysis of GTP is sufficient to release the 3′ end of the Phe-tRNA from complexation with EFTu. Translocation of the A site bound complex did not occur. The crosslink site on EFTu is probably near the periphery of the molecule, because shortening the probe from 20 Å to 14 Å completely blocked crosslinking. A similar but shorter 8 Å probe, p-azidophenacyl-4-thiouridine located on the opposite face of the tRNA, did not crosslink.
All Science Journal Classification (ASJC) codes
- Structural Biology
- Molecular Biology