Radioactive 30S and 50S subunits from E. coli ribosomes were irradiated with UV light, under conditions giving rise to approximately 10% cross-linking of protein to ribosomal RNA. Irradiation to levels of cross-linking higher than 10% caused unfolding of the ribosomal subunits, even in the presence of 5 mM magnesium. The specificity of the cross-linking reaction at this low level was found to be extremely high. Cross-linked RNA-protein complexes, freed from unbound protein, were treated with nuclease and then analysed on Sarkosyl gels. S7 was found to be the primary target of the cross-linking reaction in the 30S particle. This was proven by using subunits from both E. coli MRE 600 and A19, whose respective S7 species differ markedly. In the 50S particle, L4 was the primary target, although L2 was also cross-linked to a small extent. Ambiguity in the identification of L4 in the Sarkosyl system was resolved by two-dimensional electrophoresis, which was also used to demonstrate a genuine linkage to RNA in the case of both S7 and L4; proteins spots containing 32P were observed, derived from these two proteins, when subunits containing 32P-RNA were irradiated, treated with nuclease, and applied to the electrophoresis. The identities of S7, L4 and L2 were finally confirmed by Ouchterlony tests with protein-specific anti-sera.

Radioactive subunits from Escherichia coli ribosomes were treated with formaldehyde, with a view to inducing a controlled cross-linking reaction between protein and ribosomal RNA. Under the conditions described, about 10-15 of the total protein remained bound to the RNA in the presence of dodecyl sulphate, and little or no irreversible protein-protein cross-linking was observed. The RNA-protein complexes are of a rather unstable nature, and the reaction is spontaneously reversible.The proteins involved in the cross-linked complexes were examined by polyacrylamide gel electrophoresis, after removal of unbound protein and nuclease treatment to digest the RNA-protein complex. This showed that the individual ribosomal proteins were cross-linked to varying degrees, and most of the proteins were involved in the reaction at least to some extent. A distinct group of proteins from each subunit were, however, reproducibly cross-linked in high yield.The formaldehyde-treated subunits were subjected to a controlled hydrolysis with ribonuclease TI, and RNA fragments of known nucleotide sequence containing the cross-linked proteins were isolated. Despite the reversibility of the cross-linking reaction, sufficient cross-linked protein survived the experiment to enable an analysis to be made of proteins associated with the various RNA fragments.In the 50-S subunit, proteins L27 and L32 were found to be cross-linked to the 18-S RNA fragment which arises from the 3' end of 23-S RNA. L2, L6 and L16 were also found in this fragment in small amounts. In contrast, L13 appeared to be linked to both the 18-S RNA and the 13-S RNA fragment from the 5' end of 23-S RNA. In the 30-S subunit, proteins S3, S5, S9 (ll), S 13, and to some extent S4, S7, S 12 and S 18, were found associated with an RNA region comprising approximately 900 nucleotides at the 5' end of 16-S RNA. S4, S9 (ll), S13, and to a lesser extent S 7 and S14, were found in an RNA region comprising about 450 nucleotides near the 3' end of 16-S RNA, but lacking the 3'-terminal 150 nucleotides. Protein S21 was cross-linked to 16-S RNA, but was not found in either of the two RNA regions above.Two different biochemical approaches are currently being used to investigate RNA-protein interactions in the Escherichia coli ribosome. The first of these involves the study of complexes between isolated proteins and ribosomal RNA or fragments of ribosomal , and this method is basically intended to discover those regions of the RNA which contain stable 'binding sites' for individual proteins. The second approach involves the isolation of ribonucleoprotein fragments from ribosomal subunits, the purpose here being to investigate topographical relationships 'between proteins or groups

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