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R Brimacombe

Publications and source records attributed to R Brimacombe.

At least 109 records · Page 6Linked to original sources
PubMed1980

Precise localization of the site of cross-linking between protein L4 and 23S ribonucleic acid induced by mild ultraviolet irradiation of Escherichia coli 50S ribosomal subunits.

Mild ultraviolet irradiation of Escherichia coli 50S ribosomal subunits causes a cross-linking reaction between protein and RNA, whose primary target is protein L4 [Möller, K., & Brimacombe, R. (1975) Mol. Gen. Genet. 141, 343]. Here we have determined the site of this cross-link both on L4 and on 23S RNA. For the site on the protein, a cross-linked protein-oligonucleotide complex was isolated and subjected to successive digestions with various proteases. In each case the peptide-oligonucleotide complexes formed were analyzed. It could clearly be shown that the cross-link site was contained within a characteristic peptide 16--20 amino acids long and that the amino acid concerned was the tyrosine residue at position 35 in the recently completed L4 sequence (M. Kimura and B. Wittmann-Liebold, personal communication). For the site on the RNA, a cross-linked L4--23S RNA complex was subjected to mild nuclease digestion, producing a range of L4--RNA fragments which were isolated with the help of a new two-dimensional gel electrophoresis system. Oligonucleotide analyses of these fragments, combined with successive nuclease digestions of the residual oligonucleotide attached to protein L4, established that the site of cross-linking was homogeneous, involving a uridine residue at position 615 in the recently determined 23S RNA sequence [Brosius, J., Dull, T. J., & Noller, H. F. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 201].

Amino Acid Sequence↗
PubMed1980

An experimentally-derived model for the secondary structure of the 16S ribosomal RNA from Escherichia coli.

Ribonucleoprotein fragments are isolated by mild ribonuclease digestion of E. coli 30S ribosomal subunits, and are deproteinized and subjected to a second partial digestion. Base-pairing between the resulting small RNA fragments is investigated using the two-dimensional gel electrophoresis procedure already reported (see Ref. 1). The interactions thus found are incorporated into a secondary structure model covering approximately 80% of the 16S RNA.

Base Sequence↗
PubMed1980

Localisation of a series of intra-RNA cross-links in 16S RNA, induced by ultraviolet irradiation of Escherichia coli 30S ribosomal subunits.

Mild ultraviolet irradiation of E. coli ribosomal subunits leads to the formation of a number of intra-RNA cross-links, in addition to the RNA-protein cross-links already reported (see refs. 9, 10). After partial ribonuclease digestion of the RNA from irradiated subunits, complexes containing these intra-RNA cross-links can be isolated on a two-dimensional gel electrophoresis system, and subjected to sequence analysis. A series of these cross-linked complexes is described, and the cross-linked RNA regions are compared with the secondary structures derived for 16S RNA (see refs. 6, 7).

Base Sequence↗
PubMed1979

The use of sym-triazine trichloride in RNA-protein cross-linking studies with Escherichia coli ribosomal subunits.

The reagent sym-triazine trichloride is used as a bifunctional reagent to generate RNA-protein cross-links within intact ribosomal subunits from E. coli. The reaction takes place in a stepwise manner, involving substitution of one chlorine atom at 12 degrees and pH 8, and substitution of the second at 40 degrees and pH 6. The cross-linked proteins are analysed by two-dimensional electrophoresis, and the existence of a stable cross-linkage is demonstrated by isolating protein-oligonucleotide complexes from 32P-labelled subunits. The proteins cross-linked are S3 and S4 in the 30S subunit, and L2 in the large subunit, together with smaller amounts of other proteins. The reagent should prove useful in topographical studies of the E. coli ribosome as it is a rigid molecule and generates very short cross-links.

Electrophoresis↗
PubMed1979

Max-Planck-Institut für Molekulare Genetik, Abteilung Wittmann, Berlin-Dahlem, GFR.

It is well established that when E. coli 30S ribosomal subunits are irradiated with ultraviolet light under mild conditions a specific cross-link is formed between protein S7 and the 16S RNA. Methodology is presented for the analysis of the single nucleotide residue concerned in this cross-link. Firstly, the identity of the ribonuclease T1 octanucleotide attached to S7 is confirmed by a new method, which involves isolation and analysis of S7-polynucleotide complexes containing 30 -- 40 nucleotides. Secondly, the isolated S7-octanucleotide complex is digested successively with ribonuclease A, proteinase K and ribonuclease T2, and the nucleotides liberated are identified. The results show unambiguously that uridine residue number 1239 in the 16S RNA sequence is cross-linked to protein S7.

Base Sequence↗
PubMed1978

30S ribosomal proteins cross-linked to 16S RNA by periodate oxidation followed by borohydride reduction.

Gel electrophoretic techniques have been used to reexamine the RNA-protein cross-linking reaction induced by periodate oxidation and borohydride reduction of 30S ribosomal subunits. The results show that a number of 30S ribosomal proteins become attached to intact 16S RNA by this method, in addition to those already published. It follows that this cross-linking technique as it stands is of little value as a topographical probe of the environment of the 3'-terminus of the 16S RNA.

Bacterial Proteins↗
PubMed1978

Chemical cross-linking of protein to RNA within intact ribosomal subunits from Escherichia coli.

Bifunctional reagents, namely bis-(2-chloroethyl)-amine ("nitrogen mustard") and activated esters of 3-(2-bromo-3-oxobutane-1-sulphonyl)-propionic acid ("bromo-ketone reagent") are used to cross-linked protein to RNA within intact ribosomal subunits. The cross-linked proteins are analysed on two different two-dimensional gel electrophoresis sytems, and the existence of a stable cross-linkage is demonstrated by isolating cross-linked protein-oligonucleotide complexes from subunits containing 32P-labelled RNA. Proteins S3, S4, S5, S9/S11 and S13 from the 30S subunit, and proteins L1 and L2 from the 50S subunit were cross-linked to RNA by the nitrogen mustard, together with a number of other so far unresolved proteins. Correspondingly S3, S4, S7, S9/S11 and L12 were cross-linked by the bromoketone reagent, although in lower yield. The reagents should prove useful topographical studies on ribosomal subunits, and arguments are presented favouring the use of non-cleavable and relatively non-specific RNA-protein cross-linking reagents for such studies.

Butanones↗
PubMed1978

Application of a rapid gel method to the sequencing of fragments of 16S ribosomal RNA from Escherichia coli.

A gel sequencing method has been applied to two 5' end-labelled fragments of the 16S ribosomal RNA from E. coli. The procedure involves partial enzymatic hydrolysis by ribonucleases T1, U2 or A, in order to generate series of end-labelled subfragments terminating in guanine, adenine, or pyrimidine residues, respectively. The two fragments concerned were approximately 75 and 90 nucleotides in length, and both arose from the 3' region of the 16S RNA. The sequences deduced are compared with the published sequence of 16S RNA, and contribute information to the final ordering of the ribonuclease T1 oligonucleotides in the latter, as well as revealing some probable errors.

Base Sequence↗
PubMed1978

RNA-protein cross-linking in Eschericia coli 30S ribosomal subunits: a method for the direct analysis of the RNA regions involved in the cross-links.

A prerequisite for topographical studies on ribosomal subunits involving RNA-protein cross-linking is that the cross-linking sites on the RNA should be determined. Methodology is presented which offers a solution to this problem, using as a test system 30S subunits in which protein S7 has been cross-linked to the 16S RNA by ultraviolet irradiation. The method is based on a gel separation system in the presence of a non-ionic detergent. When a ribonucleoprotein fragment containing RNA-protein cross-links is applied to this system, non-cross-linked protein is removed, and simultaneously the cross-linked RNA-protein complex is separated from non-cross-linked RNA. Oligonucleotide analysis of the S7-RNA complex isolated in this manner showed it to consist of a region of RNA from sections P-A of the 16S RNA. A single characteristic oligonucleotide was absent from this region, and it was tentatively concluded that this missing oligonucleotide contains the actual site of cross-linking.

Bacterial Proteins↗
PubMed1978

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

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 cross-linking 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.

Escherichia coli↗
PubMed1978

Ribosome structure.

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Amino Acid Sequence↗