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Biomedical subjects

R Brimacombe

Publications and source records attributed to R Brimacombe.

At least 73 records · Page 4Linked to original sources
PubMed1990

Selective isolation and detailed analysis of intra-RNA cross-links induced in the large ribosomal subunit of E. coli: a model for the tertiary structure of the tRNA binding domain in 23S RNA.

Intramolecular RNA cross-links were induced within the large ribosomal subunit of E. coli 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 two-dimensional 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 1782 and 2608-2609, 1940 and 2554, 1941-1942 and 1964-1965, 1955 and 2552-2553, 2145-2146 and 2202, 2518-2519 and 2544-2545, and between positions 2790-2791 and 2892-2895 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.

Base Sequence↗
PubMed1989

Site-directed cross-linking of mRNA analogues to the Escherichia coli ribosome; identification of 30S ribosomal components that can be cross-linked to the mRNA at various points 5' with respect to the decoding site.

Three different mRNA analogues (28 to 34 nucleotides long) were prepared by T7 transcription from synthetic DNA templates. Each message contained the sequence ACC-GCG (coding for threonine and alanine, respectively), together with a single thio-U residue located at a variable position on the 5'-side of these coding triplets. A photo-reactive group was introduced by substitution of the thio-U with 4-azidophenacyl bromide. The messages were bound to E. coli 70S ribosomes in the presence of the appropriate tRNA-Thr or tRNA-Ala, and the azidophenyl group was photoactivated. Cross-linking was found to occur exclusively within the 30S subunit, with the 32P-label in the cross-linked mRNA being divided roughly equally between 30S ribosomal proteins and 16S RNA. Immunological analysis of the cross-linked proteins showed that, in the presence of either tRNA species, protein S7 was the primary target, whereas in the absence of tRNA only small amounts of protein S21 were cross-linked. The cross-link site to 16S RNA lay in all cases very close to its extreme 3'-terminus. These data indicate that the outgoing message leaves the cleft of the 30S subunit in a "northerly" direction.

Base Sequence↗
PubMed1988

Intra-RNA cross-linking in Escherichia coli 30S ribosomal subunits: selective isolation of cross-linked products by hybridization to specific cDNA fragments.

M13 clones were constructed with cDNA inserts corresponding to specific regions of E. coli ribosomal RNA. The DNA from the clones was immobilized by coupling to diazobenzyloxymethyl cellulose, and was used for the selective isolation by hybridization of cross-linked RNA complexes containing the complementary sequences. Immobilized DNA samples with inserts complementary to four different regions covering bases 735-1384 of the 16S RNA were hybridized with a mixture of 16S RNA fragments generated by partial digestion of 30S subunits that had been cross-linked by ultraviolet irradiation in vivo. After dehybridization, the individual RNA fragments and cross-linked complexes were separated by gel electrophoresis and analysed by our usual procedures. Nine cross-links are described; four of these are hitherto unobserved "secondary structural" cross-links, and one is a new "tertiary structural" cross-link between positions 243-247 and 891-894 of the 16S RNA.

Base Sequence↗
PubMed1988

Covalent cross-linking of poly(A) to Escherichia coli ribosomes, and localization of the cross-link site within the 16S RNA.

Poly(A) can be cross-linked to E. coli 70S ribosomes in the presence of tRNALys by mild ultraviolet irradiation. The cross-linking reaction is exclusively with the 30S subunit, and involves primarily the RNA moiety. Following a partial nuclease digestion, cross-linked complexes containing poly(A) and fragments of the 16S RNA were isolated by affinity chromatography on oligo(dT)-cellulose. The complexes were purified by gel electrophoresis and subjected to oligonucleotide analysis, which revealed a single cross-link site within positions 1394-1399 of the 16S RNA. The same pattern of cross-linking, at about one-fifth of the intensity, was observed in the absence of tRNALys. The cross-link site to poly(A), together with other sites in the 16S RNA that have been implicated in ribosomal function, is discussed in the framework of our recent model for the three-dimensional structure of 16S RNA; all of the functional sites are clustered together in two distinct groups in the model.

Chromatography, Affinity↗
PubMed1988

Protein binding sites on Escherichia coli 16S ribosomal RNA; RNA regions that are protected by proteins S7, S9 and S19, and by proteins S8, S15 and S17.

Selected groups of isolated 14C-labelled proteins from E. coli 30S ribosomal subunits were reconstituted with 32P-labelled 16S RNA, and the reconstituted complexes were partially digested with ribonuclease A. RNA fragments protected by the proteins were separated by gel electrophoresis and subjected to sequence analysis. Complexes containing proteins S7 and S19 protected an RNA region comprising helices 29 to 32, part of helix 41, and helices 42 and 43 of the 16S RNA secondary structure. Addition of protein S9 had no effect. When compared with previous data for proteins S7, S9, S14 and S19, these results suggest that S14 interacts with helix 33, and that S9 and S14 together interact with the loop-end of helix 41. Complexes containing proteins S8, S15 and S17 protected helices 7 to 10 as well as the "S8-S15 binding site" (helices 20, 22 and parts of helices 21 and 23). When protein S15 was omitted, S8 and S18 showed protection of part of helix 44 in addition to the latter regions. The results are discussed in terms of our model for the detailed arrangement of proteins and RNA in the 30S subunit.

Base Sequence↗
PubMed1988

RNA-protein cross-linking in Escherichia coli 50S ribosomal subunits; determination of sites on 23S RNA that are cross-linked to proteins L2, L4, L24 and L27 by treatment with 2-iminothiolane.

RNA-protein cross-links were introduced into E. coli 50S ribosomal subunits by treatment with 2-iminothiolane followed by mild ultraviolet irradiation. After partial digestion of the RNA, the cross-linked RNA-protein complexes were separated by our recently published three-step procedure. In cases where this separation was inadequate, a further purification step was introduced, involving affinity chromatography with antibodies to the ribosomal 50S proteins. Analysis of the isolated complexes enabled four new cross-link sites on the 23S RNA to be identified, as well as re-confirming several previously established sites. The new sites are as follows: Protein L2 is cross-linked within an oligonucleotide at positions 1818-1823 in the 23S RNA, protein L4 within positions 320-325, protein L24 within positions 99-107, and protein L27 within positions 2320-2323.

Base Sequence↗
PubMed1988

A detailed model of the three-dimensional structure of Escherichia coli 16 S ribosomal RNA in situ in the 30 S subunit.

A large body of intra-RNA and RNA-protein crosslinking data, obtained in this laboratory, was used to fold the phylogenetically and experimentally established secondary structure of Escherichia coli 16 S RNA into a three-dimensional model. All the crosslinks were induced in intact 30 S subunits (or in some cases in growing E. coli cells), and the sites of crosslinking were precisely localized on the RNA by oligonucleotide analysis. The RNA-protein crosslinking data (including 28 sites, and involving 13 of the 21 30S ribosomal were used to relate the RNA structure to the distribution of the proteins as determined by neutron scattering. The three-dimensional model of the 16 S RNA has overall dimensions of 220 A x 140 A x 90 A, in good agreement with electron microscopic estimates for the 30 S subunit. The shape of the model is also recognizably the same as that seen in electron micrographs, and the positions in the model of bases localized on the 30 S subunit by immunoelectron microscopy (the 5' and 3' termini, the m7G and m6(2)A residues, and C-1400) correspond closely to their experimentally observed positions. The distances between the RNA-protein crosslink sites in the model correlate well with the distances between protein centres of mass obtained by neutron scattering, only two out of 66 distances falling outside the expected tolerance limits. These two distances both involve protein S13, a protein noted for its anomalous behaviour. A comparison with other experimental information not specifically used in deriving the model shows that it fits well with published data on RNA-protein binding sites, mutation sites on the RNA causing resistance to antibiotics, tertiary interactions in the RNA, and a potential secondary structural "switch". Of the sites on 16 S RNA that have been found to be accessible to chemical modification in the 30 S subunit, 87% are at obviously exposed positions in the model. In contrast, 70% of the sites corresponding to positions that have ribose 2'-O-methylations in the eukaryotic 18 S RNA from Xenopus laevis are at non-exposed (i.e. internal) positions in the model. All nine of the modified bases in the E. coli 16 S RNA itself show a remarkable distribution, in that they form a "necklace" in one plane around the "throat" of the subunit. Insertions in eukaryotic 18 S RNA, and corresponding deletions in chloroplast or mammalian mitochondrial ribosomal RNA relative to E. coli 16 S RNA represent distinct sub-domains in the structure.(ABSTRACT TRUNCATED AT 400 WORDS)

Computer Graphics↗
PubMed1988

The Escherichia coli 30S ribosomal subunit; an optimized three-dimensional fit between the ribosomal proteins and the 16S RNA.

We have generated a computerized fit between the 3-dimensional map of the E.coli 30S ribosomal proteins, as determined by neutron scattering, and the recently published 3-dimensional model for the 16S RNA. To achieve this, the framework of coordinates for RNA-protein cross-link sites on the phosphate backbone in the RNA model was related to the corresponding framework of coordinates for the mass centres of the proteins by a least squares fitting procedure. The resulting structure, displayed on a computer graphics system, gives the first complete picture of the E.coli 30S ribosomal subunit showing both the proteins and the double-helical regions of the RNA. The root mean square distance between cross-link sites and protein centres is 32 A. The position of the mass centre of the combined double-helical regions was calculated from the model and compared with the position of the mass centre of the complete set of proteins. The two centres are displaced relative to one another by 20 A in the model structure, in good agreement with the experimental value of 25 A found by neutron scattering.

Computer Graphics↗
PubMed1988

A model for the spatial arrangement of the proteins in the large subunit of the Escherichia coli ribosome.

A three-dimensional model for the arrangement of 29 of the 33 proteins from the Escherichia coli large ribosomal subunit has been generated by interactive computer graphics. The topographical information that served as input in the model building process was obtained by combining the immunoelectron microscopically determined network of epitope-epitope distances on the surface of the large ribosomal subunit with in situ protein-protein cross-linking data. These two independent sets of data were shown to be compatible by geometric analysis, thus allowing the construction of an inherently consistent model. The model shows (i) that the lower third of the large subunit is protein-poor, (ii) that proteins known to be functionally involved in peptide bond formation and translocation are clustered in two separate regions, (iii) that proteins functionally interdependent during the self-assembly of the large subunit are close neighbours in the mature subunit and (iv) that proteins forming the early assembly nucleus are grouped together in a distinct region at the 'back' of the subunit.

Bacterial Proteins↗
PubMed1987

A rapid immunological spot test for the identification of proteins in covalently linked protein-nucleic acid complexes.

A method is described for the rapid immunological identification of proteins in studies of multicomponent systems. In this case the system is the E. coli ribosome, and the ribosomal proteins to be identified are covalently attached to fragments of labelled ribosomal RNA as a result of chemical cross-linking procedures. Antisera raised against the individual ribosomal proteins are spotted onto a nitrocellulose sheet, and an aliquot of the covalent complex under test is added to each antibody spot. After suitable washing procedures, a positive reaction with one or other of the antisera is visualized by autoradiography of the labelled RNA moiety attached to the antibody via the ribosomal protein. Amounts of protein as low as 10 pg can readily be detected.

Cross-Linking Reagents↗
PubMed1987

Protein binding sites on Escherichia coli 16S RNA; RNA regions that are protected by proteins S7, S14 and S19 in the presence or absence of protein S9.

14C-labelled proteins from E. coli 30S ribosomal subunits were isolated by HPLC, and selected groups of these proteins were reconstituted with 32P-labelled 16S RNA. The isolated reconstituted particles were partially digested with ribonuclease A, and the RNA fragments protected by the proteins were separated by gel electrophoresis and subjected to sequence analysis. Protein S7 alone gave no protected fragments, but S7 together with S14 and S19 protected an RNA region comprising the sequences 936-965, 972-1030, 1208-1262 and 1285-1379 of the 16S RNA. Addition of increasing amounts of protein S9 to the S7/S14/S19 particle resulted in a parallel increase in the protection of the hairpin loop between bases 1262 and 1285. The results are discussed in terms of the three-dimensional folding of 16S RNA in the 30S subunit.

Bacterial Proteins↗
PubMed1987

RNA-protein cross-linking in Escherichia coli 30S ribosomal subunits; determination of sites on 16S RNA that are cross-linked to proteins S3, S4, S5, S7, S8, S9, S11, S13, S19 and S21 by treatment with methyl p-azidophenyl acetimidate.

RNA-protein cross-links were introduced into E. coli 30S ribosomal subunits by treatment with methyl p-azidophenyl acetimidate. After partial nuclease digestion of the RNA moiety, a number of cross-linked RNA-protein complexes were isolated by a new three-step procedure. Protein and RNA analysis of the individual complexes gave the following results: Proteins S3, S4, S5 and S8 are cross-linked to the 5'-terminal tetranucleotide of 16S RNA. S5 is also cross-linked to the 16S RNA within an oligonucleotide encompassing positions 559-561. Proteins S11, S9, S19 and S7 are cross-linked to 16S RNA within oligonucleotides encompassing positions 702-705, 1130-1131, 1223-1231 and 1238-1240, respectively. Protein S13 is cross-linked to an oligonucleotide encompassing positions 1337-1338, and is also involved in an anomalous cross-link within positions 189-191. Protein S21 is cross-linked to the 3'-terminal dodecanucleotide of the 16S RNA.

Bacterial Proteins↗
PubMed1987

RNA-protein cross-linking in Escherichia coli 30S ribosomal subunits; determination of sites on 16S RNA that are cross-linked to proteins S3, S4, S7, S9, S10, S11, S17, S18 and S21 by treatment with bis-(2-chloroethyl)-methylamine.

RNA-protein cross-links were introduced into E. coli 30S ribosomal subunits by treatment with bis-(2-chloroethyl)-methylamine. After partial nuclease digestion of the RNA moiety, a number of cross-linked RNA-protein complexes were isolated by a new three-step procedure. Protein and RNA analysis of the individual complexes gave the following results: proteins S4 and S9 are cross-linked to the 16S RNA at positions 413 and 954, respectively. Proteins S11 and S21 are both cross-linked to the RNA within an oligonucleotide encompassing positions 693-697, and proteins S17, S10, S3 and S7 are cross-linked within oligonucleotides encompassing positions 278-280, 1139-1144, 1155-1158, and 1531-1542, respectively. A cross-link to protein S18 was found by a process of elimination to lie between positions 845 and 851.

Bacterial Proteins↗
PubMed1986

Investigation of the tertiary folding of Escherichia coli ribosomal RNA by intra-RNA cross-linking in vivo.

"In vivo" cross-links were introduced into ribosomal RNA by direct ultraviolet irradiation of intact Escherichia coli cells, during growth in a 32P-labelled medium. Ribosomes were isolated from the irradiated cultures, dissociated into subunits and subjected to partial digestion with cobra venom nuclease. The intra-RNA cross-linked fragments were separated by two-dimensional gel electrophoresis and the sites of cross-linking determined, using our published methodology. A comparison with the data previously obtained by this procedure, after irradiation of isolated 30 S and 50 S subunits, showed that in the case of the 50 S subunit nine out of the ten previous cross-links in the 23 S RNA could be identified in the "in vivo" experiments, and correspondingly in the 30 S subunit five out of the six previous cross-links in the 16 S RNA were identified. Some new cross-links were found, as well as two cross-links in the 16 S RNA, which had hitherto only been observed after partial digestion of irradiated 30 S subunits with ribonuclease T1. The relevance of these data to the tertiary folding of the rRNA in situ is discussed, with particular reference to the work of other authors, in which "naked" RNA was used as the substrate for cross-linking and model-building studies.

Escherichia coli↗
PubMed1986

The ribosomal binding domain of the Escherichia coli release factors. Modification of tyrosine in the N-terminal domain of ribosomal protein L11 affects release factors 1 and 2 differentially.

Ribosomal protein L11 is one of only two ribosomal proteins significantly iodinated when Escherichia coli 50 S subunits are modified by immobilized lactoperoxidase, and the major target has been shown previously to be tyrosine at position 7 in the N-terminal domain. This modification reduces in vitro termination activity with release factor (RF)-1 by 70-90%, but RF-2 activity is less affected (30-50%). The loss of activity parallels incorporation of iodine into the subunit. The 50 S subunits from L11-lacking strains of bacteria have highly elevated activity with RF-2 and low activity with RF-1. The iodination does not affect RF-2 activity but reduces the RF-1 activity further. Ribosomal proteins, L2, L6, and L25, are significantly labeled in L11-lacking ribosomes in contrast to the control 50 S subunits. L11 has been modified in isolation and incorporated back efficiently into L11-lacking ribosomes. This L11, iodinated also predominantly at Tyr 7, is unable to restore RF-1 activity to L11-lacking ribosomes in contrast to mock-iodinated protein. These results suggest the involvement of the N terminus of L11 in the binding domain of the bacterial release factors and indicate that there are subtle differences in how the two factors interact with the ribosome.

Binding Sites↗