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

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Abstract
Mild UV irradiation of E. coli 50S ribosomal subunits causes a cross-linking reaction between protein and RNA, whose primary target is protein L4. The site of this cross-link both on L4 and on 23S RNA was determined. 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. 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 2-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 cross-linking was homogeneous, involving a uridine residue at position 615 in the recently determined 23S RNA sequence.

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