Ribosomal RNA‐protein cross‐links, induced by γ‐irradiation of 40S and 60S ribosomal subunits of L cells

Abstract

The 40s and 60s ribosomal subunits of L cells are y-irradiated in the absence of oxygen at low radiation doses to keep the integrity of the ribosomal structure. We show that under these experimental conditions, specific cross-links are induced in situ between rRNA and ribosomal proteins due to close contact between their reactive groups.We found that about 15 proteins are cross-linked to the 28s RNA. Most of them belong to the core proteins of the 60s ribosomal subunits. A few high-molecular mass proteins which might be factors are also bound to 28s RNA.Between 8 and 11 proteins are covalently linked to 18s RNA; 8 of these have been previously described as transferable proteins from one subunit to the other. Only 3 are core proteins of the small subunit.The contribution of these results to the study of the three-dimensional ribosomal structure is also discussed.The proteins and nucleic acids in ribosomes are arranged in a specific three-dimensional configuration to form structural and functional domains. Several studies have been made to identify the proteins that interact with the different species of MgSO,. Buffer I11 : 10 mM phosphate buffer pH 7.4, 0.5 mM MgSO,, 200 mM LiCl, 0.1 % sodium dodecylsulfate (SDS).Buffer IV: 10mM phosphate buffer pH 7.4, 100mM LiC1. rRNA~in eukaryotes [l, 21. During the last few years, covalent cross-linking techniques have been introduced for investigating the structural organisation of the ribosomes. Chemical reagents have been used in order to identify the binding sites for initiation factors [3,4] and to obtain information on the protein topography of both subunits [5 -71. Ultraviolet irradiation has been found to produce RNA-protein cross-links [8]. All these data are only now being integrated in terms of the ribosomal three-dimensional structure. Recently, y radiations appeared as an original and valuable tool to induce highly specific RNAprotein covalent bonds in Escherichia coli ribosomes [9,10]. At low doses, they reach the reactive targets in situ, while preserving the structure. Only a few chemical groups are capable of forming radicals and these reactive groups must be very close to form a covalent link [9, lo]. In order to gain more insight on the eukaryotic ribosomal structure, we applied, in this study, the y radiation technique to 40s and 60s ribosomal subunits of L cells. The proteins found cross-linked to rRNA have been identified among the distinct classes of ribosomal proteins defined in a previous study: those split off by KC1, core, transferable and labelled proteins [l 11.These results are discussed in a topological approach to the relationship of the RNA and the protein in the structure of the two ribosomal subunits. MATERIALS AND METHODS BuffersBuffer I contains 10 mM phosphate buffer pH 7.4, 5 mM MgSO,. Buffer I1 : 10 mM phosphate buffer pH 7.4, 0.5 mM Abbreviation. SDS, sodium dodecyl sulfate. Cell growth and labelling conditionsMouse C1 I D LM (TK-) cells were grown in suspension in an Eagle-Dulbecco medium supplemented with 10 % calf serum [12]. For la...

Photo‐induced protein cross‐linking to 5S RNA and 28–5.8S RNA within rat‐liver 60S ribosomal subunits

“…The fact that numerous proteins were coss-linked to 28s RNA is in conformity with the results of different authors who used 60s rat liver ribosomal subunits treated with either 1-ethyl-3-dimethylaminopropyl carbodiimide or diepoxybutane (for references see Table l), or employed y-irradiated 60s ribosomal subunits of L cells [24] (we did not mention this work in Table 1 because the proteins have not been numbered with the uniform nomenclature). Our 5 S + 28 -5.8 S RNA-protein cross-linking results (Table 1) also confirm to a large extent those we previously obtained using an indirect method [7].…”

Structure and function of ribosomal RNA

“…The methods and reagents that have been applied to RNA-protein cross-linking in the ribosome, as well as the problems associated with this approach, were reviewed in detail recently , and so will only be treated briefly here. Since our above-mentioned review, Brewer & Noller (1983) have described a new cross-linking reagent which is a bis-kethoxal derivative, and the use of y-irradiation for inducing RNA-protein cross-links has also been reported (Cazillis et al, 1984). However, since it is already clear that most if not all of the ribosomal proteins are capable of being cross-linked to their cognate RNA molecules in situ in the ribosomal subunits by one reagent or another, the question of determining the sites of cross-linking to individual proteins is now the dominant problem.…”