Photochemical crosslinking unmodified acetylvalyl-tRNA to 16S RNA at the ribosomal P site

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Abstract
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 [peptidyl] site as judged by reactivity with puromycin. Subsequent irradiation (> 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. tRNA whose 4-thiouridine were modified with unreactive analogs of p-azidophenacyl bromide or unmodified acetylvalyl-tRNA exhibited the same cross-linking behavior as photoaffinity probe-modified acetylvalyl-tRNA. Even acetylvalyl-tRNA whose 4-thiouridine was removed by treatment with H2O2 was quantitatively as active as unmodified tRNA. These results provide the 1st demonstration of direct photochemical cross-linking of tRNA to ribosomes. [Escherichia coli were used.].

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