New aspects of the eukaryotic ribosomal subunit interaction (original) (raw)
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Interaction of 5-S RNA, 5.8-S RNA and tRNA with Rat-Liver Ribosomal Proteins
European Journal of Biochemistry, 1978
In order to ascertain 5-S RNA, 5.8-S RNA and tRNA binding proteins from eukaryotic ribosomes, affinity chromatography of the rat liver ribosomal 40-S and 60-S subunit proteins on immobilized RNAs was used. Rat liver 5-S RNA, 5.8-S RNA and tRNA were immobilized via 3 '-end ribose to adipic-acid-hydrazide-epoxy-activated Sepharose 6B. Using two-dimensional polyacrylamide gel electrophoresis in a urea-urea system, the bound proteins were identified as follows : a) 5-S RNA was found to form a complex containing mainly proteins L6 and L18 of the large ribosomal subunit, whereas proteins L7, L8 and L35 were present in the complex to a lesser degree.
Structure of the ribosome-associated 5ยท 8 S ribosomal RNA
Journal of molecular biology, 1983
The structure of the 5-8 S ribosomal RNA in rat liver ribosomes was probed by comparing dimethyl sulfate-reactive sites in whole ribosomes, 60 S subunits, the 5.8 S-28 S rRNA complex and the free 5"8 S rRNA under conditions of salt and temperature that permit protein synthesis in vitro. Differences in reactive sites between the free and both the 28 S rRNA and 60S subunit-associated 5-8 S rRNA show that significant conformational changes occur when the molecule interacts with its cognate 28 S rRNA and as the complex is further integrated into the ribosomal structure. These results indicate that, as previously suggested by phylogenetic comparisons of the secondary structure, only the "G+C-rich" stem may remain unaltered and a universal structure is probably present only in the whole ribosome or 60S subunit. Further comparisons with the ribosomeassociated molecule indicate that while the 5.8 S rRNA may be partly localized in the ribosomal interface, four cytidylic acid residues, Cs6, C10o, Ct2 ~ and C12s, remain reactive even in whole ribosomes. In contrast, the cytidylic acid residues in the 5 S rRNA are not accessible in either the 60 S subunit or the intact ribosome. The nature of the structural rearrangements and potential sites of interaction with the 28 S rRNA and ribosomal proteins are discussed. Although the 5.8 S rRNA of eukaryotic ribosomes was identified over a decade ago (Pene et al., 1968; Weinberg & Penman, 1968), little is yet known about this molecule's role in ribosome structure or function. At least two suggestions for its function, i.e. roles in transfer RNA binding (Nishikawa & Takemura, 1974; Wrede & Erdmann, 1977) or subunit interaction (Ulbrich et al., 1979; Toots et al., 1979), have been postulated but these are entirely based on indirect evidence and must be tested experimentally before any conclusions are reached. Recently, as a possible step towards direct information about the molecule's role in the ribosome, we examined the topography of this RNA in ribosomes and ribosomal subunits using diethyl pyrocarbonate reactivity as a probe (Lo & Nazar, 1981,1982). The results suggested a number of interesting features, including conformational changes and the possibility that part of the molecule may be within the ribosomal interface. The study, however, had two major limitations: only regions containing adenylic and certain guanylic acid residues could be probed and artifacts resulting from diethyl pyrocarbonate reactivity with proteins could not be ruled out entirely. To overcome these limitations, a parallel study has been conducted using a different chemical probe, dimethyl sulfate. The results strongly support the conclusions drawn earlier, but describe new regions of accessibility and further indicate that the changes in the ribosomeassociated molecule are considerably more extensive than previously believed. Active ribosomes were prepared from male Wistar rat livers essentially as described by Rendi & Hultin (1960) and subunits were prepared from the purified ribosomes using the methods of Terao & Ogata (1970) with minor modifications
European Journal of Biochemistry, 1984
The ribonucleoprotein complex between 5-S RNA and its binding protein (5-S RNA . protein complex) of yeast ribosomes was released from 60-S subunits with 25 mM EDTA and the protein component was purified by chromatography on DEAE-cellulose. This protein, designated YL3 (MI = 36000 on dodecylsulfate gels), was relatively insoluble in neutral solutions (pH 4-9) and migrated as one of four acidic 60-S subunit proteins when analyzed by the Kaltschmidt and Wittman two-dimensional gel system. Amino acid analyses indicated lower amounts of lysine and arginine than most ribosomal proteins. Sequence homology was observed in the N terminus of YL3, and two prokaryotic 5-S RNA binding proteins, EL 18 from Escherichia coliand HL 13 from Halobacterium cutirubrum : Ala'-
Role of 5S RNA in the Functions of 50S Ribosomal Subunits
Proceedings of the National Academy of Sciences, 1971
50S ribosomal subunits from Bacillus stearothermophilus can be reconstituted from their dissociated components, namely a 5S RNA-free protein fraction, a 5S RNA-free 23S ribosomal RNA fraction, and purified 5S RNA. The biological activity of reconstituted particles in polypeptide synthesis is dependent on the presence of 5S RNA. In the absence of 5S RNA, particles are produced that have greatly reduced activity in ( a ) polypeptide synthesis directed by synthetic, as well as natural, messenger RNA, ( b ) peptidyl transferase assay, ( c ) [ 3 H]UAA binding dependent on peptide chain termination factor R1, ( d ) G factor-dependent [ 3 H]GTP binding, and ( e ) codon-directed tRNA binding assayed in the presence of 30S subunits. Thus, 5S RNA is an essential 50S ribosomal component.
Binding sites of ribosomal proteins on prokaryotic 5S RNAs: a study with ribonucleases
Biochemistry, 1982
The binding sites of ribosomal proteins L18 and L25 on 5s RNA from Escherichia coli were probed with ribonucleases A, TI, and T2 and a double helix specific cobra venom endonuclease. The results for the protein-RNA complexes, which were compared with those for the free RNA [Douthwaite, S., & Garrett, R. A. (1981) Biochemistry 20, 7301-73071, reveal an extensive interaction site for protein L18 and a more localized one for L25. Generally comparable results, with a few important differences, were obtained in a study of the binding sites of the two E. coli proteins on Bacillus stearothermophilus 5s RNA. Several protein-induced changes in the RNA structures were identified; some are
Functional Roles of 50-S Ribosomal Proteins
European Journal of Biochemistry, 1977
Ribosomal proteins previously inactivated by treatment with fluorescein isothiocyanate have been incorporated into 50-S ribosomal subunits during reconstitution from particles disassembled by 2 M LiCl in the presence of an excess of the modified proteins. The reconstituted particles show alterations in some functional activities resulting from the incorporation of the inactive ribosomal proteins added exogenously. Of the fluorescein-isothiocyanate-treated proteins incorporated, L24 and L25 drastically affect all the activities tested and these proteins possibly play a fundamental role in determining the overall structure of the particle. Proteins L16 and LIO are apparently involved both in the GTP hydrolysis dependent on elongation factor G and in peptidyl transferase activity but the modified protein L11 only affects GTPase activity indirectly and interferes with the ribosome assembly process involving proteins L7 and L12. Protein L1 may be involved with peptidyl transferase activity while proteins L7 and L12, in agreement with many reports in the literature, affect the factor-dependent hydrolysis of GTP.
Journal of Biological Chemistry, 1982
The topography of 5.8 S rRNA in rat liver ribosomes has been examined by comparing diethyl pyrocarbonate-reactive sites in free 5.8 S RNA, the 5.8 5-28 S rRNA complex, 60 S subunits, and whole ribosomes. The ribosomal components were treated with diethyl pyrocarbonate under salt and temperature conditions which allow cell-free protein synthesis; the 5.8 S rRNA was extracted, labeled in vitro, chemically cleaved with aniline, and the fragments were analyzed by rapid gelsequencing techniques. Differences in the cleavage patterns of free and 28 S or ribosome-associated 5.8 S rRNA suggest that conformational changes occur when this molecule is assembled into ribosomes. In whole ribosomes, the reactive sites were largely restricted to the ttAU-rich" stem and an increased reactivity at some of the nucleotides suggested that a major change occurs in this region when the RNA interacts with ribosomal proteins. The reactivity was generally much less restricted in 60 S subunits but increased reactivity in some residues was also observed. The results further indicate that in rat ribosomes, the two-G-A-A-C-sequences, putative binding sites for tRNA, are accessible in 60 S subunits but not in whole ribosomes and suggest that part of the molecule may be located in the ribosomal interface. When compared to 5 S rRNA, the free 5.8 S RNA molecule appears to be generally more reactive with diethyl pyrocarbonate and the cleavage patterns suggest that the 5 S RNA molecule is completely restricted or buried in whole ribosomes. Although our knowledge of the structure of ribosomal RNAs is growing rapidly, the elucidation of their functional roles in protein synthesis continues to be a formidable problem. A variety of studies indicate that the 3'-end of the single 16-18 S RNA component in the small ribosomal subunit is probably involved in mRNA binding (1) and several functional roles also have been suggested for the 5 S rRNA component including tRNA binding (2), a GTPase activity (3), and subunit interaction (4,5). There is little solid evidence to support any of these roles and it seems unlikely that all of these roles reside in this one small RNA molecule. Virtually nothing is currently known about the function of the 23-28 S rRNA component of the large ribosomal subunit or the eukaryotic 5.8 S rRNA. Recently, however, several lines of speculation have suggested that the 5.8 S RNA molecule may replace in part the 5 S RNA in its tRNA-binding role (6, 7). The 5.8 S rRNA, which is hydrogen-bonded to its cognate * This work was supported by Grant A6666 of the Medical Research Council of Canada. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.