Mild ultraviolet irradiation of Escherichia coli 50S ribosomal subunits causes a cross-linking 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 cross-linking was homogeneous, involving a uridine residue at position 615 in the recently determined 23S RNA sequence [Brosius, J., Dull, T. J., & Noller, H. F. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 201].

Bifunctional reagents, namely bis-(2-chloroethyl)-amine ("nitrogen mustard") and activated esters of 3-(2-bromo-3-oxobutane-1-sulphonyl)-propionic acid ("bromo-ketone reagent") are used to cross-linked protein to RNA within intact ribosomal subunits. The cross-linked proteins are analysed on two different two-dimensional gel electrophoresis sytems, and the existence of a stable cross-linkage is demonstrated by isolating cross-linked protein-oligonucleotide complexes from subunits containing 32P-labelled RNA. Proteins S3, S4, S5, S9/S11 and S13 from the 30S subunit, and proteins L1 and L2 from the 50S subunit were cross-linked to RNA by the nitrogen mustard, together with a number of other so far unresolved proteins. Correspondingly S3, S4, S7, S9/S11 and L12 were cross-linked by the bromoketone reagent, although in lower yield. The reagents should prove useful topographical studies on ribosomal subunits, and arguments are presented favouring the use of non-cleavable and relatively non-specific RNA-protein cross-linking reagents for such studies.

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