Precise Localization of the Site of Crosslinking between Protein L4 and 23S Ribonucleic Acid Induced by Mild Ultraviolet Irradiation of Escherichia Coli 50S Ribosomal Subunits

Mild ultraviolet irradiation of Escherichia coli 50S ribosomal subunits causes a crosslinking reaction between protein and RNA, whose primary target is protein L4 [Möller, K., & Brimacombe, R. (1975)>Mol. Gen. Genet. 141, 343], Here we have determined the site of this cross-link both on L4 and on 23S RNA. For the site on the protein, a cross-linked protein-oligonucleotide complex was isolated and subjected to successive digestions with various proteases. In each case the peptide-oligonucleotide complexes formed were analyzed. It could clearly be shown that the cross-link site was contained within a characteristic peptide 16-20 amino acids long and that the amino acid concerned was the tyrosine residue at position 35 in the recently completed L4 sequence (M. Kimura and B. Wittmann-Liebold, personal communication). For the site on the RNA, a cross-linked L4-23S RNA complex was subjected to mild nuclease digestion, producing a range of L4-RNA fragments which were isolated with the help of a new two-dimensional gel electrophoresis system. Oligonucleotide analyses of these fragments, combined with successive nuclease digestions of the residual oligonucleotide attached to protein L4, established that the site of crosslinking was homogeneous, involving a uridine residue at position 615 in the recently determined 23S RNA sequence.