Identification of intermolecular RNA cross-links at the subunit interface of the Escherichia coli ribosome (original) (raw)
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Evidence for RNA-RNA cross-link formation in Escherichia coli ribosomes
Nucleic Acids Research - NAR, 1978
Evidence is presented in three separate cases for the formation of RNA-RNA cross-links in intact E. coli ribosomes and ribosomal subunits. The first case is a cross-link between the 18S and 13S regions of the 23S RNA, induced by ultraviolet irradiation. The second is a cross-link at the subunit interface, generated by the bifunctional reagent bis-(2-chloroethyl)-amine. The third example is a cross-link between sections O'-D and P-A of the 16S RNA, induced as in the first case by ultraviolet irradiation. The RNA-RNA cross-links can be identified as such, despite the complications introduced by concomitant RNA-protein crosslinking reactions. The experiments represent a first attempt to introduce RNA-RNA cross-linking into studies of the topographical organization of the RNA within the ribosome.
Nucleic Acids Research, 1983
RNA-protein cross-links were introduced into E. coli 30S ribosomal subunits by reaction with 2-iminothiolane followed by a mild ultraviolet irradiation treatment. After removal of nonreacted protein and partial nuclease digestion of the crosslinked 16S RNA-protein moiety, a number of individual crosslinked complexes could be isolated and the sites of attachment of the proteins to the RNA determined. Protein S8 was crosslinked to the RNA at three different positions, within oligonucleotides encompassing positions 629-633, 651-654, and (tentatively) 593-597 in the 16S sequence. Protein S7 was crosslinked within two oligonucleotides encompassing positions 1238-1240, and 1377-1378. In addition, a site at position 723-724 was observed, cross-linked to protein S19, S20 or S21.
Journal of Molecular Biology, 1978
When 30 S ribosomal subunits are irradiated with ultraviolet light, we have found that) an RNA-protein crosslinking reaction occurs whose primary target, is protein S7. This paper describes the identification of the oligopeptide and oligonucleotide at the crosslinking point, by direct analysis (a) of the peptide remaining attached t,o an oligonucleotide (after total digestion of the RNA in the crosslinked complex with ribonucleases A and T1, followed by digestion with trypsin), and (b) of the nucleotidesremaining attached to the crosslinked protein (aft,er digestion of the RNA in the complex with ribonuclease T, alone). The crosslinking site was found to lie within a single short peptide, Ser-Met-Ala-Leu-Arg (posit'ions 113 to 117 in the 87 sequence), with methionine as thta probable amino acid concerned. The principal RNA site was found to lie within an oligonucleotide three to six bases long, the underlined portion of the partially ordered sequence C-U-A-C-A-A-U-G.G.C-G in section I' of the 16 S RNA. The methodology involved has been designed with a view to being generally applicable in future RNA-protein crosslinking studies, where several proteins arc simultaneously attached to the RNA.
Nucleic Acids Research, 1989
Functionally active 70S ribosomes containing 4-thiouridine (s4U in place of uridine were prepared by a formerly described in vivo substitution method. Proteins were crosslinked to RNA by 366nm photoactivation of s"U. We observe the systematic and caracteristic formation of 305 dimers; they were eliminated for analysis of RNA-protein crosslinks. M13 probes containing rDNA inserts complementary to domains 1 and 2 of 16S RNA from the 5'end up to nucleotide 868 were used to select contiguous or overlapping RNA sections. The proteins covalently crosslinked to each RNA section were identified as 53. 54, 55. 57, 59, S18, S20 and S21. Several crosslinks are compatible with previously published sites for proteins S5, 518, 520 and 521 ; others for proteins 53, 54, 57, 59, 518 correspond necessarily to new sites. 1475 Nucleic Acids Research Volume 17 Number 4 1989 Nucleic Acids Research covalent crosslinked complexes. As a general and systematic approach to obtain low resolution mapping of crosslinked 305 proteins on 165 RNA. We have used single stranded Mi 3 DNA probes (ssDNA) containing rDNA inserts complementary to 16S RNA sections. These probes were hybridized to 1 6S RNA-protein complexes and RNase Ti hydrolysis was used to eliminate the non selected regions. The proteins covalently linked to the RNA sections protected by the complementary rDNA were identified by two-dimensional gel analysis. This method was previously described in details for domains 3 and 4 (12-14). We report here the use of 8 other M13 ssDNA probes to identify proteins crosslinked to sections between nucleotides 1 and 869, covering domains 1 and 2. We have found several RNA-protein crosslinks which are compatible with previous results and a few others that are necessarily new sites. MATERIAL AND METHODS. Material and Enzymes. 4-thiouridine was from Sigma. (1251) and the T4 DNA ligase were from Amersham (England). Carrier free (H932P04) was from CEA (France). The Klenow fragment or DNA polymerase was from Boehringer and RNAse T 1, RNase T2 from Sankyo. Buffers. Buffer A: 10 mM triethanolamine / HCl pH 7.5, 1 mM Mg AC2, 50 mM KCl Buffer B: 1OmM triethanolamine / HC1 pH 7.5; 0.1% SDS, 100 mM LICI. Buffer SCE I X: 15mM sodium citrate pH 7, 150mM NaCl, 1OmM Na2 EDTA. Buffer TBE 1X: 90mM Tris, 90mM borate , 2.5mM Na2 EDTA pH 8.3. Cell strains and media used for ribosome DreDaration.
Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression, 1990
A large number of intra-RNA and RNA-Iwotein cross-link sites have been localized within the 238 RNA from E. co//50 S ribosomal subunits. These sites, tngetlmr with other data, are sufficient to constrain the secondary ~ of the 23 S molecule into a compact three-dimensional shape. Some of the features of this s~ are discussed, in particular, those relating to the orientation of tRNA on the 50 S subunit as studied by site-directed cross-linking tedmklueS. A correspemling model for the 168 RNA within the 30 S subunit has already been described, and here a site-directed cross-linking altC¢oaeh is being used to determine the path followed through the sulmnit by messenger RNA. 0167-4781/90/$03.50
Comparative cross-linking study on the 50S ribosomal subunit from Escherichia coli
Biochemistry, 1989
We have carried out an extensive protein-protein cross-linking study on the 50s ribosomal subunit of Escherichia coli using four different cross-linking reagents of varying length and specificity. For the unambiguous identification of the members of the cross-linked protein complexes, immunoblotting techniques using antisera specific for each individual ribosomal protein have been used, and for each cross-link, the cross-linking yield has been determined. With the smallest cross-linking reagent diepoxybutane (4 A), four cross-links have been identified, namely, L3-Ll9, L10-L11, L13-L21, and L14-LI9. With the sulfhydryl-specific cross-linking reagent o-phenylenedimaleimide (5.2 A) and p-phenylenedimaleimide (1 2 A),
Nucleic Acids Research, 1990
Intramolecular RNA cross-links were induced within the large ribosomal subunit of E. coil by mild ultraviolet irradiation. Regions of the 23S RNA previously implicated in interactions with ribosomal-bound tRNA were then specifically excised by addressed cleavage using ribonuclease H, in conjunction with synthetic complementary decadeoxyribonucleotides. Individual cross-linked fragments within these regions released by such 'directed digests' were isolated by twodimensional gel electrophoresis and the sites involved in the cross-links determined using classical oligonucleotide analysis techniques. Using this approach, seven 'new' cross-links could be precisely localised, between positions in the 23S RNA sequence. These data, in conjunction with data from RNA-protein cross-linking studies carried out in our laboratory, were used to define a model for the tertiary organisation of the tRNA binding domain of 23S RNA 'in situ', in which the specific nucleotides associated with tRNA binding in the 'A' and 'P' sites are clustered at the base of the 'central protuberance' of the 50S subunit.