Direct contacts between 16-S RNA and split proteins S2, S3, S 5 , S14 and S21 inside the 30-S subunit of Eschevichia coli ribosomes were evidenced by the formation of ultraviolet-induced (A = 254 nni) RNA-protein cross-links. 30-S subunits were reassembled from core particles and a mixture of split proteins containing in each case a single '251-labelled protein. All the proteins tested are cross-linked as a result of a single-hit process; proteins S3 and S21 were cross-linked at the highest rate.The essential step in the study of nucleoproteins is detection of interactions between components of the complex.The evidence for direct RNA-protein contacts in ribosomes of Eschevichia coli was presented for a number of proteins [l -31. They belong mostly to a group of proteins which do not readily dissociate from 16-S RNA upon incubation of 30-S subparticles at high concentrations of LiCl or CsCl and which form specific RNA . protein complexes (core proteins) [4,5]. At the same time, there was no evidence for the existence of direct RNA-protein contacts in the 30-S subparticle for a number of so-called split proteins that readily dissociate from the ribosome at high concentrations of LiCl or CsCl and do not form strong complexes with 16-S RNA.Only recently [6] Hochkeppel and Craven showed that some split proteins are able to bind independently to 16-S RNA prepared by acetic acid urea extraction. They presented some evidence that six of these proteins (S3, S5, S9, S11, S12 and SlS) bind site-specifically to such isolated 16-S RNA. But the stability and specificity of nucleoproteins are conditioned by cooperative interactions. Therefore the data, obtained for incomplete complexes, seem not to be very conclusive.For detection of contacts between a number of split proteins (S2, S3, S5, S14 and S21) and 16-S RNA in the complete 30-S subunit of E. coli ribosomes we have used ultraviolet-induced cross-links. This method is adequate for fixation of polynucleotideprotein contacts in intact nucleoproteins [7,8] including ribosomes [9-121. 30-S subunits were reconstituted from a mixture of core subparticles and split proteins, containing in each case a single "'Ilabelled protein component. The reconstituted subunits were irradiated with ultraviolet light (i = 253.7 nm, 2.5-25 x lo5 quanta per subparticle) and the formation of cross-links was monitored by analysis of cosedimentation of 16-S RNA and the labelled protein in the presence of sodium dodecyl sulphate and EDTA.It was shown that proteins S3 and S21 cross-linked well with 16-S RNA, whereas proteins S2, S5 and S14 cross-linked under the same conditions to a much lesser extent.
MATERIALS AND METHODS30-S subunits of E. coli MRE 600 ribosomes were prepared by zonal centrifugation [I 31. Individual ribosomal proteins were isolated according to [14]. 100-300 pg of each isolated protein were labelled with 1-3 pCi of Na'251 (Amersham, 18 mCi/pg) by the method of Ball et al. [15] with a slight modification [16]. The purity of the labelled proteins was checked by gel electrophoresis...