Biomedical subjects

R Brimacombe

Publications and source records attributed to R Brimacombe.

At least 19 recordsLinked to original sources

PubMed1999

The environment of 5S rRNA in the ribosome: cross-links to 23S rRNA from sites within helices II and III of the 5S molecule.

Three contiguous fragments of Escherichia coli 5S rRNA were prepared by T7 transcription from synthetic DNA templates. The central fragment, comprising residues 33-71 of the molecule, was transcribed in the presence of 4-thiouridine triphosphate together with [32P]UTP. The three transcripts were ligated together, yielding a 5S rRNA analogue carrying 4-thiouridine residues at positions 40, 48, 55 and 65 in helices II and III. After ligation, the 4-thiouridine residues were derivatised with p -azidophenacyl bromide. The modified 5S rRNA was reconstituted into 50S subunits and these subunits were used to prepare 70S ribosomes in the presence or absence of tRNA and mRNA. The azidophenyl groups were then photoactivated by mild irradiation at 300 nm and the products of cross-linking analysed by our standard procedures. Multiple cross-links from 5S rRNA to two distinct regions of the 23S rRNA were observed. The first region was located in helix 38 in Domain II of the 23S molecule, with cross-links at sites between nucleotides 885 and 922. The second region covered helices 81-85 in Domain V, with sites between nucleotides 2272 and 2345. Taken together with previous data, these results serve to define the arrangement of the 5S rRNA molecule relative to the 23S rRNA within the 50S subunit.

Bacteriophage T7↗

PubMed1999

The Database of Ribosomal Cross-links: an update.

The Database of Ribosomal Cross-links (DRC) was created in 1997. Here we describe new data incorporated into this database and several new features of the DRC. The DRC is freely available via World Wide Web at http://visitweb.com/database/ or http://www. mpimg-berlin-dahlem.mpg.de/ approximately ag_ribo/ag_brimacombe/drc/

Bacterial Proteins↗

PubMed1998

Flexibility of the nascent polypeptide chain within the ribosome--contacts from the peptide N-terminus to a specific region of the 30S subunit.

The ribosomal environment of the N-terminus of the nascent polypeptide chain has been investigated using peptides of different lengths, synthesized in situ on Escherichia coli ribosomes; the peptides each carry a photoreactive diazirine moiety at their N-terminus, so as to generate cross-links to neighbouring ribosomal components. Our previous studies [Choi, K. M. & Brimacombe, R. (1998) Nucleic Acids Res. 26, 887-895] with three independent families of peptides, derived from the E. coli ompA protein gene, the tetracycline-resistance gene and the bacteriophage T4 gene 60, identified a series of sites within the 23S rRNA to which the peptides became cross-linked. The distribution of these cross-links indicated that the nascent peptide is very flexible within the 50S subunit. Here, we demonstrate that the N-termini of the ompA and gene-60 peptides can, in addition, even become concomitantly cross-linked to the 30S subunit. The cross-linking is predominantly to 30S ribosomal proteins S1, S2, S4 and (to a lesser extent) S3, which form a cluster near to the decoding region. This result is discussed in terms of the flexibility of the nascent peptide during the co-translational folding process, and in terms of the 'ribosomal bypass' phenomenon which is known to occur during translation of the gene 60 mRNA.

Amino Acid Sequence↗

PubMed1998

Correlation of the expansion segments in mammalian rRNA with the fine structure of the 80 S ribosome; a cryoelectron microscopic reconstruction of the rabbit reticulocyte ribosome at 21 A resolution.

Samples of 80 S ribosomes from rabbit reticulocytes were subjected to electron cryomicroscopy combined with angular reconstitution. A three-dimensional reconstruction at 21 A resolution was obtained, which was compared with the corresponding (previously published) reconstruction of Escherichia coli 70 S ribosomes carrying tRNAs at the A and P sites. In the region of the intersubunit cavity, the principal features observed in the 70 S ribosome (such as the L1 protuberance, the central protuberance and A site finger in the large subunit) could all be clearly identified in the 80 S particle. On the other hand, significant additional features were observed in the 80 S ribosomes on the solvent sides and lower regions of both subunits. In the case of the small (40 S) subunit, the most prominent additions are two extensions at the base of the particle. By comparing the secondary structure of the rabbit 18 S rRNA with our model for the three-dimensional arrangement of E. coli 16 S rRNA, these two extensions could be correlated with the rabbit expansion segments (each totalling ca 170 bases) in the regions of helix 21, and of helices 8, 9 and 44, respectively. A similar comparison of the secondary structures of mammalian 28 S rRNA and E. coli 23 S rRNA, combined with preliminary modelling studies on the 23 S rRNA within the 50 S subunit, enabled the additional features in the 60 S subunit to be sub-divided into five groups. The first (corresponding to a total of ca 335 extra bases in helices 45, 98 and 101) is located on the solvent side of the 60 S subunit, close to the L7/L12 area. The second (820 bases in helices 25 and 38) is centrally placed on the solvent side of the subunit, whereas the third group (totaling 225 bases in helices 18/19, 27/29, 52 and 54) lies towards the L1 side of the subunit. The fourth feature (80 bases in helices 78 and 79) lies within or close to the L1 protuberance itself, and the fifth (560 bases in helix 63) is located underneath the L1 protuberance on the interface side of the 60 S subunit.

Animals↗

PubMed1998

The environment of 5S rRNA in the ribosome: cross-links to the GTPase-associated area of 23S rRNA.

Two photoreactive diazirine derivatives of uridine were used to study contacts between 5S rRNA and 23 rRNA in situ in Escherichia coli ribosomes. 2'-Amino-2'-deoxy-uridine or 5-methyleneaminouridine were introduced into 5S rRNA by T7 transcription. After incorporation of these uridine analogues into the transcript their amino groups were modified with 4-[3-(trifluoromethyl)-3 H -diazirin-3-yl]benzyl isothiocyanate or the N -hydroxysuccinimide ester of 4-[3-(trifluoromethyl)-3 H -diazirin-3-yl]benzoic acid respectively. 5S rRNA carrying the photoreactive diazirine groups (referred to as the 2'-aminoribose derivative and the 5-methyleneamino derivative respectively) was reconstituted into 50S subunits or 70S ribosomes. After mild UV irradiation cross-links formed to 23S rRNA were analysed by standard procedures. All of the observed cross-links involved residue U89 of the 5S rRNA. Three nucleotides of 23S rRNA were cross-linked to this residue with the 5-methyleneamino derivative, namely U958, G1022 and G1138. With the 2'-aminoribose derivative a single cross-link was found, to U958. The significance of these cross-links for our understanding of the structure and function of 5S rRNA and its environment in the ribosome are discussed.

Base Sequence↗

PubMed1998

The path of the growing peptide chain through the 23S rRNA in the 50S ribosomal subunit; a comparative cross-linking study with three different peptide families.

As part of a programme to investigate the path of the nascent peptide through the large ribosomal subunit, peptides of different lengths (up to 30 amino acids), corresponding to the signal peptide sequence and N-terminal region of the Escherichia coli ompA protein, were synthesized in situ on E.coli ribosomes. The peptides each carried a diazirine moiety attached to their N-terminus which, after peptide synthesis, was photoactivated so as to induce cross-links to the 23S rRNA. The results showed that, with increasing length, the peptides became progressively cross-linked to sites in Domains V, II, III and I of the 23S rRNA, in a similar manner to that previously observed with a family of peptides derived from the tetracycline resistance gene. However, the cross-links to Domain III appeared at a shorter peptide length (12 aa) in the case of the ompA sequence, and an additional cross-link in Domain II (localized to nt 780-835) was also observed from this peptide. As with the tetracycline resistance sequence, peptides of all lengths were still able to form cross-links from their N-termini to the peptidyl transferase centre in Domain V. A further set of peptides (30 or 50 aa long), derived from mutants of the bacteriophage T4 gene 60 sequence, did not show the cross-links to Domain III, but their N-termini were nevertheless cross-linked to Domain I and to the sites in Domains II and V. The ability of relatively long peptides to fold back towards the peptidyl transferase centre thus appears to be a general phenomenon.

Amino Acid Sequence↗

PubMed1998

The Database of Ribosomal Cross links (DRC).

The Database of Ribosomal Cross-links (DRC) provides a complete collection of all the published data produced by cross-linking studies on the Escherichia coli ribosome, as well as on its components and functional ligands. The DRC currently includes data on 986 cross-links from >100 research papers, yielded by >40 different reagents. For each cross-link, information is given concerning its location in the ribosome, the chemical or photochemical reagent applied, a brief description of the method(s) used to locate the cross-link, and the literature reference. The DRC is freely available via the World Wide Web at: http://Ribosome.Genebee.MSU.SU/DRC/ or at http://WWW:MPIMG-Berlin-Dahlem.MPG.DE/[symbol: see text]baranov/DRC/

Computer Communication Networks↗

PubMed1998

Matching the crystallographic structure of ribosomal protein S7 to a three-dimensional model of the 16S ribosomal RNA.

Two recently published but independently derived structures, namely the X-ray crystallographic structure of ribosomal protein S7 and the "binding pocket" for this protein in a three-dimensional model of the 16S rRNA, have been correlated with one another. The known rRNA-protein interactions for S7 include a minimum binding site, a number of footprint sites, and two RNA-protein crosslink sites on the 16S rRNA, all of which form a compact group in the published 16S rRNA model (despite the fact that these interactions were not used as primary modeling constraints in building that model). The amino acids in protein S7 that are involved in the two crosslinks to 16S rRNA have also been determined in previous studies, and here we have used these sites to orient the crystallographic structure of S7 relative to its rRNA binding pocket. Some minor alterations were made to the rRNA model to improve the fit. In the resulting structure, the principal positively charged surface of the protein is in contact with the 16S rRNA, and all of the RNA-protein interaction data are satisfied. The quality of the fit gives added confidence as to the validity of the 16S rRNA model. Protein S7 is furthermore known to be crosslinked both to P site-bound tRNA and to mRNA at positions upstream of the P site codon; the matched S7-16S rRNA structure makes a prediction as to the location of this crosslink site within the protein molecule.

Anticodon↗

PubMed1998

New features of 23S ribosomal RNA folding: the long helix 41-42 makes a "U-turn" inside the ribosome.

23S rRNA from Escherichia coli was cleaved at single internucleotide bonds using ribonuclease H in the presence of appropriate chimeric oligonucleotides; the individual cleavage sites were between residues 384 and 385, 867 and 868, 1045 and 1046, and 2510 and 2511, with an additional fortuitous cleavage at positions 1117 and 1118. In each case, the 3' terminus of the 5' fragment was ligated to radioactively labeled 4-thiouridine 5'-,3'-biphosphate ("psUp"), and the cleaved 23S rRNA carrying this label was reconstituted into 50S subunits. The 50S subunits were able to associate normally with 30S subunits to form 70S ribosomes. Intra-RNA crosslinks from the 4-thiouridine residues were induced by irradiation at 350 nm, and the crosslink sites within the 23S rRNA were analyzed. The rRNA molecules carrying psUp at positions 867 and 1117 showed crosslinks to nearby positions on the opposite strand of the same double helix where the cleavage was located, and no crosslinking was detected from position 2510. In contrast, the rRNA carrying psUp at position 384 showed crosslinking to nt 420 (and sometimes also to 416 and 425) in the neighboring helix in 23S rRNA, and the rRNA with psUp at position 1045 gave a crosslink to residue 993. The latter crosslink demonstrates that the long helix 41-42 of the 23S rRNA (which carries the region associated with GTPase activity) must double back on itself, forming a "U-turn" in the ribosome. This result is discussed in terms of the topography of the GTPase region in the 50S subunit, and its relation to the locations of the 5S rRNA and the peptidyl transferase center.

Base Sequence↗

PubMed1997

Visualization of elongation factor Tu on the Escherichia coli ribosome.

The delivery of a specific amino acid to the translating ribosome is fundamental to protein synthesis. The binding of aminoacyl-transfer RNA to the ribosome is catalysed by the elongation factor Tu (EF-Tu). The elongation factor, the aminoacyl-tRNA and GTP form a stable 'ternary' complex that binds to the ribosome. We have used electron cryomicroscopy and angular reconstitution to visualize directly the kirromycin-stalled ternary complex in the A site of the 70S ribosome of Escherichia coli. Electron cryomicroscopy had previously given detailed ribosomal structures at 25 and 23 A resolution, and was used to determine the position of tRNAs on the ribosome. In particular, the structures of pre-translocational (tRNAs in A and P sites) and post-translocational ribosomes (P and E sites occupied) were both visualized at a resolution of approximately 20 A. Our three-dimensional reconstruction at 18 A resolution shows the ternary complex spanning the inter-subunit space with the acceptor domain of the tRNA reaching into the decoding centre. Domain 1 (the G domain) of the EF-Tu is bound both to the L7/L12 stalk and to the 50S body underneath the stalk, whereas domain 2 is oriented towards the S12 region on the 30S subunit.

Crystallography, X-Ray↗

PubMed1997

A new model for the three-dimensional folding of Escherichia coli 16 S ribosomal RNA. I. Fitting the RNA to a 3D electron microscopic map at 20 A.

Recently published models of the Escherichia coli 70 S ribosome at 20 A resolution, obtained by cryo-electron microscopy (cryo-EM) combined with computerized image processing techniques, exhibit two features that are directly relevant to the in situ three-dimensional folding of the rRNA molecules. First, at this level of resolution many fine structural details are visible, a number of them having dimensions comparable to those of nucleic acid helices. Second, in reconstructions of ribosomes in the pre- and post-translocational states, density can be seen that corresponds directly to the A and P site tRNAs, and to the P and E site tRNAs, respectively, thus enabling the decoding region on the 30 S subunit to be located rather precisely. Accordingly, we have refined our previous model for the 16 S rRNA, based on biochemical evidence, by fitting it to the cryo-EM contour of ribosomes carrying A and P site tRNAs. For this purpose, the most immediately relevant evidence consists of new site-directed cross-linking data in the decoding region, which define sets of contacts between the 16 S rRNA and mRNA, or between 16 S rRNA and tRNA at the A, P and E sites; these contact sites can be correlated directly with the tRNA positions in the EM structure. The model is extended to other parts of the 16 S molecule by fitting individual elements of the well-established secondary structure of the 16 S rRNA into the appropriate fine structural elements of the EM contour, at the same time taking into account other data used in the previous model, such as intra-RNA cross-links within the 16 S rRNA itself. The large body of available RNA-protein cross-linking and foot-printing data is also considered in the model, in order to correlate the rRNA folding with the known distribution of the 30 S ribosomal proteins as determined by neutron scattering and immuno-electron microscopy. The great majority of the biochemical data points involve single-stranded regions of the rRNA, and therefore, in contrast to most previous models, the single-stranded regions are included in our structure, with the help of a specially developed modelling programme, ERNA-3D. This allows the various biochemical data sets to be displayed directly, in this and in the accompanying papers, on diagrams of appropriate parts of the rRNA structure within the cryo-EM contour.

Base Sequence↗

PubMed1997

A new model for the three-dimensional folding of Escherichia coli 16 S ribosomal RNA. II. The RNA-protein interaction data.

The map of the mass centres of the 21 proteins from the Escherichia coli 30 S ribosomal subunit, as determined by neutron scattering, was fitted to a cryoelectron microscopic (cryo-EM) model at a resolution of 20 A of 70 S ribosomes in the pre-translocational state, carrying tRNA molecules at the A and P sites. The fit to the 30 S moiety of the 70 S particles was accomplished with the help of the well-known distribution of the ribosomal proteins in the head, body and side lobe regions of the 30 S subunit, as determined by immuno electron microscopy (IEM). Most of the protein mass centres were found to lie close to the surface (or even outside) of the cryo-EM contour of the 30 S subunit, supporting the idea that the ribosomal proteins are arranged peripherally around the rRNA. The ribosomal protein distribution was then compared with the corresponding model for the 16 S rRNA, fitted to the same EM contour (described in an accompanying paper), in order to analyse the mutual compatibility of the arrangement of proteins and rRNA in terms of the available RNA-protein interaction data. The information taken into account included the hydroxyl radical and base foot-printing data from Noller's laboratory, and our own in situ cross-linking results. Proteins S1 and S14 were not considered, due to the lack of RNA-protein data. Among the 19 proteins analysed, 12 (namely S2, S4, S5, S7, S8, S9, S10, S11, S12, S15, S17 and S21) showed a fit to the rRNA model that varied from being excellent to at least acceptable. Of the remaining 7, S3 and S13 showed a rather poor fit, as did S18 (which is considered in combination with S6 in the foot-printing experiments). S16 was difficult to evaluate, as the foot-print data for this protein cover a large area of the rRNA. S19 and S20 showed a bad fit in terms of the neutron map, but their foot-print and cross-link sites were clustered into compact groups in the rRNA model in those regions of the 30 S subunit where these proteins have respectively been located by IEM studies.

Base Sequence↗

PubMed1997

A new model for the three-dimensional folding of Escherichia coli 16 S ribosomal RNA. III. The topography of the functional centre.

We describe the locations of sites within the 3D model for the 16 S rRNA (described in two accompanying papers) that are implicated in ribosomal function. The relevant experimental data originate from many laboratories and include sites of foot-printing, cross-linking or mutagenesis for various functional ligands. A number of the sites were themselves used as constraints in building the 16 S model. (1) The foot-print sites for A site tRNA are all clustered around the anticodon stem-loop of the tRNA; there is no "allosteric" site. (2) The foot-print sites for P site tRNA that are essential for P site binding are similarly clustered around the P site anticodon stem-loop. The foot-print sites in 16 S rRNA helices 23 and 24 are, however, remote from the P site tRNA. (3) Cross-link sites from specific nucleotides within the anticodon loops of A or P site-bound tRNA are mostly in agreement with the model, whereas those from nucleotides in the elbow region of the tRNA (which also exhibit extensive cross-linking to the 50 S subunit) are more widely spread. Again, cross-links to helix 23 are remote from the tRNAs. (4) The corresponding cross-links from E site tRNA are predominantly in helix 23, and these agree with the model. Electron microscopy data are presented, suggestive of substantial conformational changes in this region of the ribosome. (5) Foot-prints for IF-3 in helices 23 and 24 are at a position with close contact to the 50 S subunit. (6) Foot-prints from IF-1 form a cluster around the anticodon stem-loop of A site tRNA, as do also the sites on 16 S rRNA that have been implicated in termination. (7) Foot-print sites and mutations relating to streptomycin form a compact group on one side of the A site anticodon loop, with the corresponding sites for spectinomycin on the other side. (8) Site-specific cross-links from mRNA (which were instrumental in constructing the 16 S model) fit well both in the upstream and downstream regions of the mRNA, and indicate that the incoming mRNA passes through the well-defined "hole" at the head-body junction of the 30 S subunit.

Base Sequence↗

PubMed1997

A new technique for the characterization of long-range tertiary contacts in large RNA molecules: insertion of a photolabel at a selected position in 16S rRNA within the Escherichia coli ribosome.

A new approach for inserting a photo-label at a selected position within the long ribosomal RNA molecules has been developed. The Escherichia coli 16S rRNA was cleaved at a single internucleotide bond, 1141-1142, with RNase H in the presence of a complementary chimeric oligonucleotide. 4-Thiouridine 5', 3'-diphosphate was ligated to the 3'-end of the 5'fragment at the cleavage site with T4 RNA ligase. The 16S rRNA fragments containing this added photo-reactive nucleotide were assembled together with total 30S ribosomal proteins into small ribosomal subunits. The ability of such 30S particles containing fragmented rRNA to form 70S ribosomes has been demonstrated previously. Crosslinks were induced within the 30S subunits by mild UV irradiation. The sites of crosslinking within the 16S rRNA were then analyzed using RNase H digestion and reverse transcription. Two crosslinks from the thio-nucleotide attached to nt C1141 of 16S rRNA were observed, namely to nt U1295 and G1272. These results are in agreement with the established proximity of helix 39 and 41 in the 3D structure of the 30S ribosomal subunit, as shown by other intra RNA crosslinking data. These data furthermore allow us to refine the structural arrangement of helices 41 and 39 relative to one another.

Bacteriophage T4↗

PubMed1997

Identification and sequence analysis of contact sites between ribosomal proteins and rRNA in Escherichia coli 30 S subunits by a new approach using matrix-assisted laser desorption/ionization-mass spectrometry combined with N-terminal microsequencing.

Cross-linked peptide-oligoribonucleotide complexes derived from distinct regions of the rRNA and individual ribosomal proteins of the 30 S ribosomal subunits from Escherichia coli were isolated and purified. Cross-linking sites at the amino acid and nucleotide level were determined by N-terminal amino acid sequence analysis in combination with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). MALDI-MS analysis performed subsequent to a partial alkaline hydrolysis of cross-linked peptide-oligoribonucleotide complexes allowed for the first time the cross-linked rRNA moiety to be sequenced by this technique. In this manner Lys-44 in S4 was determined to be cross-linked to the oligoribonucleotide at positions 1531-1542 on the 16 S RNA (whereby either U-1541 or A-1542 is the actual cross-link site), Lys-75 in S7 to positions 1374-1379 (C-1378 cross-linked), Met-114 in S7 to 1234-1241 (U-1240 cross-linked), Lys-55 in S8 to 651-654 (U-653 cross-linked), and Lys-29 in S17 to 629-633 (U-632 cross-linked). The novel approach applied here promises to be useful for similar studies on other known protein.RNA complexes.

Amino Acid Sequence↗

PubMed1997

Arrangement of tRNAs in pre- and posttranslocational ribosomes revealed by electron cryomicroscopy.

The three-dimensional structure of the translating 70S E. coli ribosome is presented in its two main conformations: the pretranslocational and the posttranslocational states. Using electron cryomicroscopy and angular reconstitution, structures at 20 A resolution were obtained, which, when compared with our earlier reconstruction of "empty" ribosomes, showed densities corresponding to tRNA molecules--at the P and E sites for posttranslocational ribosomes and at the A and P sites for pretranslocational ribosomes. The P-site tRNA lies directly above the bridge connecting the two ribosomal subunits, with the A-site tRNA fitted snugly against it at an angle of approximately 50 degrees, toward the L7/L12 side of the ribosome. The E-site tRNA appears to lie between the side lobe of the 30S subunit and the L1 protuberance.

Binding Sites↗

PubMed1997

The path of mRNA through the bacterial ribosome: a site-directed crosslinking study using new photoreactive derivatives of guanosine and uridine.

Two new photoreactive nucleotide derivatives have been applied in site-directed crosslinking studies with mRNA analogues. 6-Thioguanosine triphosphate or 5-methyleneaminouridine triphosphate was incorporated into mRNA analogues by T7 transcription; after transcription, the 5-methyleneaminouridine residues were converted to a diazirine derivative. mRNA analogues carrying either 6-thioguanosine or the diazirine derivative were bound to Escherichia coli ribosomes in the presence of tRNA(f)(Met), and photo-crosslinking was induced by irradiation at 350 nm. With 6-thioguanosine, specific crosslinks were observed from downstream positions +8 or +9 of the mRNA to nt 1196 in helix 34 of the 16S rRNA, and from position +12 to nt 530 in helix 18. With the diazirine derivative, a crosslink from position +2 (within the AUG codon) to nt 926 in helix 28 was found. Taken together with previous data obtained from downstream sites in mRNA analogues carrying 4-thiouridine residues, specific crosslinks have now been identified from downstream mRNA positions +2, +4, +6, +7, +8, +9, +11, and +12. The data confirm that the three 16S rRNA regions involved-helices 18, 28, and 34-are in the direct neighborhood of the decoding area of the 30S subunit.

Bacteriophage T7↗

PubMed1997

Decoding the translational termination signal: the polypeptide chain release factor in Escherichia coli crosslinks to the base following the stop codon.

Protein release factors act like tRNA analogues in decoding translational stop signals. Statistical analysis of the sequences at translational stop sites and functional studies with particular signals indicate this mimicry involves an increase in the length of the signal in the mRNA. The base following the stop codon (+4 base) is of particular interest because it has a strong influence on the competitiveness of the stop signal at recoding sites, suggesting it might form part of the release factor recognition element. Site-directed crosslinking from the +4 base showed that it is in close proximity to the Escherichia coli release factor-2 in a termination complex, a prerequisite for the +4 base being part of the recognition element. Fingerprinting analysis indicates that crosslinking to the release factor occurred from both +1 and +4 positions of the stop signal in the same RNA molecule. This provides more evidence that the +4 base may be an integral part of the decoding signature in the mRNA during the termination phase of protein biosynthesis.

Base Composition↗