A 16S rRNA-tRNA product containing a nucleotide phototrimer and specific for tRNA in the P/E hybrid state in the Escherichia coli ribosome

Ribosome complexes containing deacyl-tRNA(1)(Val) or biotinylvalyl-tRNA(1)(Val) and an mRNA analog have been irradiated with wavelengths specific for activation of the cmo(5)U nucleoside at position 34 in the tRNA(1)(Val) anticodon loop. The major product for both types of tRNA is the cross-link between 16S rRNA (C1400) and the tRNA (cmo(5)U34) characterized already by Ofengand and his collaborators [Prince et al. (1982) Proc. Natl Acad. Sci. USA, 79, 5450-5454]. However, in complexes containing deacyl-tRNA(1)(Val), an additional product is separated by denaturing PAGE and this is shown to involve C1400 and m(5)C967 of 16S rRNA and cmo(5)U34 of the tRNA. Puromycin treatment of the biotinylvalyl-tRNA(1)(Val) -70S complex followed by irradiation, results in the appearance of the unusual photoproduct, which indicates an immediate change in the tRNA interaction with the ribosome after peptide transfer. These results indicate an altered interaction between the tRNA anticodon and the 30S subunit for the tRNA in the P/E hybrid state compared with its interaction in the classic P/P state.