covalent crosslinking of trna1val to 16s rna at the ribosomal p site identification of crosslinked residues nacetylvalyltrna1val acvaltrna1val was bound to the p site of uniformly 32plabeled 70s ribosomes from escherichia coli and crosslinked to 16s rna in the 30s ribosomal subunit by irradiation with light of 300400 nm to identify the crosslinked nucleotide in 16s rna acvaltrna1val16s 32prna was digested completely with rnase t1 and the band containing the covalently attached oligonucleotides from trna and rrna was isolated by polyacrylamide gel electrophoresis the crosslinked oligonucleotide and the 32plabeled rrna moiety released from it by photoreversal of the crosslink at 254 nm were then analyzed by secondary hydrolysis with pancreatic rnase a and rnase u2 the oligonucleotide derived from 16s rna was found to be the evolutionarily conserved sequence uacacaccg1401 and the nucleotide crosslinked to trna1val c1400 the identity of the covalently attached residue in the trna was established by using acvaltrna1val16s rna prepared from unlabeled ribosomes this complex was digested to completion with rnase t1 and the resulting rna fragments were labeled at the 3 end with 532ppcp the crosslinked t1 oligonucleotide isolated from the mixture yielded one major endlabeled component upon photoreversal chemical sequence analysis demonstrated that this product was derived from the anticodoncontaining pentadecanucleotide of trna1val caccucccucmo5uacm6aag39cmo5u 5carboxymethoxyuridine a similar study of the crosslinked oligonucleotide revealed that the residue covalently bound to 16s was cmo5u34 the 5 or wobble base of the anticodon the adduct is believed to result from formation of a cyclobutane dimer between cmo5u34 of trna1val and c1400 of the 16s rna