Polynucleotide-Protein Interactions in the Translation System. Detection of Contacts between Some Ribosomal Split Proteins and 16-S RNA in 30-S Subunits of Escherichia coli Ribosomes

Abstract

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...

Ribosomal Protein S1 Associates with Escherichia coli Ribosomal 30‐S Subunit by Means of Protein‐Protein Interactions

“…(0) Radioactivity in irradiated samples; (0) radioactivity in non-irradiated samples; (--.-) absorbance at 254 nm. The insert shows radioactivity distribution in fractions 7 -14 plotted on an expanded scale containing '2'I-S1 and their subsequent dissociation under conditions described earlier [28], less than 1 of the total radioactivity was found in the 1 6 3 RNA fractions of the sucrose gradient (Fig. 2).…”

“…In particular this method was used to detect direct contacts between ribosomal proteins and 16-S RNA [37-391, 23-S RNA [40,41], tRNA [42] and mRNA [43] in ribosomal subunits and their respective complexes. In the case of 30-S subunits containing individual 1251-labelled proteins, ultraviolet-induced cross-links may be detected even with a quantum yield as low as lo-' by an estimation of "' 1 radioactivity in the 16-S RNA peak after dissociation and separation of ribosomal components on sucrose gradients [28].…”

“…After ultraviolet irradiation (A = 254 nm) of 30-S subunits [28] and analysed in a linear 5-20% sucrose gradient containing 25 mM Tris/ HCI, pH 7.8, 0.1 "/, sodium dodecyl sulfate, 10 mM EDTA. Sedimentation was at 4°C for 17 h a t 30000 rev./min in an SW-40 rotor.…”

“…Sl was iodinated with Na['251] (Amersham, 412 Ci/mmol) in the presence of IC1 according to Ballet al [27] with modifications [28]. To remove unbound iodine, the reaction mixture was dialysed against the binding buffer containing 15 mM Tris/HCl, pH 7.6, 7 mM MgC12, 70 mM NH4C1, 0.1 mM dithiothreitol.…”

“…To irradiated and control (nonirradiated) 30-S subunit solutions, sodium dodecyl sulfate and EDTA were added up to concentrations of 1 % and 0.02 M respectively. Then samples were analysed by sucrose gradient centrifugation as described previously [28]. In the case of S1 .…”