Two Specific Ribonucleoprotein Fragments from the 50-S Sub-Particle of Escherichia coli Ribosomes

Abstract

50-S ribosomal particles from Escherichia coli were hydrolysed with ribonuclease TI, and the ribonucleoprotein fragments produced were fractionated on polyacrylamide gel. The proteins contained in these fragments were analysed on gels containing the detergent sarkosyl (N-laurylsarcosine), using the technique already published. Two-dimensional gel electrophoresis was used for the final identification of the proteins.Two very specific fragments were obtained, the smaller containing only proteins L1 and L9, and the larger containing only L5, L18 and L25. The latter proteins have been shown by other authors to be involved in binding to 5-S RNA, but in this case the fragment contained more RNA than can be accounted for by 5-S RNA alone. This offers the possibility of determining the regions of 23-S RNA associated both with these two groups of proteins and with 5-S RNA. Both fragments are discussed in terms of a possible oligonucleotide analysis of their RNA moieties.It has been shown that under suitably controlled conditions 30-S ribosomal sub-particles from Escherichia coli can be digested with nuclease to yield specific fragments of ribonucleoprotein [l, 21. Such fragments have proved useful in several ways. Analysis of their protein content provides information as to the grouping of individual proteins within the ribosome [2], while analysis of the constituent RNA sequences can show which regions of the ribosomal RNA are associated with a particular group of proteins [3]. Further, such fragments have been used in more functionally oriented studies, such as the binding of streptomycin [l].A similar fragmentation approach has been used by other authors on the 50-S sub-particle. Allet and Spahr [4] isolated two large ribonucleoprotein fragments which showed some specificity in their protein content. Kagawa et al. [5] have reported a series of small fragments all from the 50-S sub-particle, but unfortunately were not able to described their proteins in the accepted nomenclature [6]. In this paper we describe the isolation of two small very specific ribo-The preceding paper in this series is by Pongs and Nierhaus (Mol. Gen. Genet., in press).Enzyme. Ribonuclease T, (EC 3.1.4.8).Definition. An A,,, unit is the quantity of material contained in 1 ml of a solution which has an absorbance of 1 at 260 nm, when measured in a l-cm path-length cell.-~ nucleoprotein fragments from the Escherichia coli 50-S ribosome, using the methodology we have already described for the 30-S sub-particle [2,7], extended to include two-dimensional gel electrophoresis [8] for the final identification of the proteins involved. Preliminary experiments have shown that RNA fragments of the expected order of size can be isolated from these RNA . protein complexes, suggesting that both fragments could be usefully subjected to oligonucleotide analysis. MATERIALS AND METHODS Preparation of RibosomesNH,Cl-washed ribosomal sub-particles were prepared as described previously [7], with the exception that E. coli strain A19 was used instead of MRE 600. Rib...

Evidence for RNA-RNA cross-link formation in Escherichia coli ribosomes

“…(The strong spot to the left of spot 59 is the 32P-ink used to mark the fingerprint.) print closely resembles that of the total RNA from the small ribonucleoprotein fragment "Band III" (13) , and examples of some unambiguous characteristic oligonucleotides, whose identities were confirmed where necessary by secondary digestion with ribonuclease A (see Materials and Methods) are :-spot 59 and/or 47 (sections 0' and D respectively), spot 3-11 (section 0'), spot [6][7][8][9][10][11][12][13][14][15][16] (section K), spots 38a and 29a (section P), spot 2-llb (section E) and spots 16i and 26a (section A) (see Fig. 5).…”

“…Each 20 41 aliquot was treated with 2 41 of ribonuclease A (0.1 4g/ml) for 1 hr at 25°C, and enzyme was removed by applying the solution to a 3% polyacrylamide-agarose gel containing 1 mM magnesium and 20 mM potassium, at pH 7.8 (14). The gel was run until a bromophenol blue marker had travelled 10 cm, and was then sliced and analysed for radioactivity.…”

Secondary structure of the large subunit ribosomal RNA from Escherichia coli, Zea mays chloroplast, and human and mouse mitochondrial ribosomes

“…2) and applied to two-dimensional gels, exactly as described (2,16). In other experiments, 50S subunits were dialysed against 10 mM Tris-HCl pH 7.8, 0.03 mM magnesium acetate, treated with ribonuclease T1 (12-50 units enzyme per A260 unit 50S) as described previously (17), and applied to a 5-20% polyacrylamide gradient gel containing 5 mM magnesium at pH 6 (cf. 18): This treatment releases a large number of free RNA fragments from the subunit, and regions of the gel containing these fragments (cf.…”

Structure of a cyanobacterial gene encoding the 50S ribosomal protein L9

“…Cross-linking experiments have demonstrated that the L9 protein is located in the vicinity of proteins L2 [6, 10], L10 [3] and L28 [24] in the ribosome. It is likely that this protein is also associated with proteins L1 [ 15,18], L3, L18 and L23 since these proteins are found in a small ribonucleoprotein fragment obtained from the 50S ribosomal subunit by controlled nucleolytic digestion [ 18 ].Only a few of the proteins in cyanobacterial ribosomes have been characterized. Genes for the 30S ribosomal proteins $2 $7 and S 12, namely, rps2, rps7 and rpsl2, respectively, have been cloned from Spirulinaplatensis [2,19], and the latter two have also been cloned from Anacystis nidulans [13].…”

“…Cross-linking experiments have demonstrated that the L9 protein is located in the vicinity of proteins L2 [6,10], L10 [3] and L28 [24] in the ribosome. It is likely that this protein is also associated with proteins L1 [ 15,18], L3, L18 and L23 since these proteins are found in a small ribonucleoprotein fragment obtained from the 50S ribosomal subunit by controlled nucleolytic digestion [ 18 ].…”