Biomedical subjects

R A Garrett

Publications and source records attributed to R A Garrett.

At least 73 records · Page 4Linked to original sources

PubMed1989

Protein L18 binds primarily at the junctions of helix II and internal loops A and B in Escherichia coli 5 S RNA. Implications for 5 S RNA structure.

Ribonuclease and chemical probes were used to investigate the binding sites of ribosomal protein L18 on Escherichia coli 5 S RNA using both end-labelling and reverse transcriptase procedures. The results, together with earlier data, were superimposed on a cylindrical projection of RNA double helices and most of the protection effects were found to cluster in the major groove at two sites located on one side of the RNA at the junctions of helix II with the adjoining internal loops A and B. The loop A/helix II junction was investigated using 5 S RNA mutants, produced by site-directed mutagenesis, that exhibited altered binding properties to L18. These results, together with those from a circular dichroism study of L18 complexed with the wild-type and different mutant RNAs, enabled us to assign an L18-induced conformational change to loop A. We infer that this change contributes to the co-operative binding of L5 to helix I, which may be reinforced by the binding of the very basic N-terminal peptide of L18 within the minor groove of helix I. A psoralen derivative formed a mono-addition product with U25 within loop B in the free RNA but not in the L18 complex. Moreover, the modified molecules were selected against in L18 binding experiments. Protection effects that occurred within the adjoining helix III and loop C were compatible with a tertiary interaction between loop C and loop B/helix III that could be stabilized by the L18 binding to the junction of helix II and loop B. Further support for a bipartite binding site derived from the finding that ethidium bromide molecules that are displaced from E. coli 5 S RNA by L18 intercalate both at the loop A/helix II junction and in loop B at the binding site of the psoralen derivative.

Bacterial Proteins↗

PubMed1989

Sequence, organization, transcription and evolution of RNA polymerase subunit genes from the archaebacterial extreme halophiles Halobacterium halobium and Halococcus morrhuae.

The genes for the four largest subunits, A, B', B" and C, of the DNA-dependent RNA polymerase were cloned from the extreme halophile Halobacterium halobium and sequenced and their transcription was analyzed. The downstream half of this gene cluster from another extreme halophile Halococcus morrhuae was also cloned, sequenced and its transcription products characterized. The H. halobium genes were transcribed into a common transcript from an upstream promoter in the order B", B', A and C. They are flanked by, and co-transcribed with, two smaller genes coding for 75 and 139 amino acid residues, respectively. Immediately downstream from these genes were two open reading frames that are homologous to ribosomal proteins S12 and S7 from Escherichia coli. In both extreme halophiles these genes were transcribed from their own promoter, but in Hc. morrhuae there was also considerable read-through from the RNA polymerase genes. Sequence alignment studies showed that the combined B" + B' subunits are equivalent to the B subunits of the eukaryotic polymerases I and II and to the eubacterial beta subunit, while the combined A + C subunits correspond to the A subunits of eukaryotic RNA polymerases I, II and III and to the eubacterial beta' subunit. The sequence similarity to the eukaryotic subunits was always much higher than to the eubacterial subunits. Conserved sequence regions within the individual subunits were located which are likely to constitute functionally important domains; they include sites associated with rifampicin and alpha-amanitin binding and two possible zinc binding fingers. Phylogenetic analyses based on sequence alignments confirmed that the extreme halophiles belong to the archaebacterial kingdom.

Archaea↗

PubMed1989

Antibiotic interactions at the GTPase-associated centre within Escherichia coli 23S rRNA.

A comprehensive range of chemical reagents and ribonucleases was employed to investigate the interaction of the antibiotics thiostrepton and micrococcin with the ribosomal protein L11-23S RNA complex and with the 50S subunit. Both antibiotics block processes associated with the ribosomal A-site but differ in their effects on GTP hydrolysis, which is inhibited by thiostrepton and stimulated by micrococcin. The interaction sites of both drugs were shown to occur within the nucleotide sequences A1067-A1098 within the protein L11 binding site on 23S RNA. This region of the ribosome structure is involved in elongation factor-G-dependent GTP hydrolysis and in the stringent response. No effects of drug binding were detected elsewhere in the 23S RNA. In general, the two drugs afforded 23S RNA similar protection from the chemical and nuclease probes in accord with their similar modes of action. One important exception, however, occurred at nucleotide A1067 within a terminal loop where thiostrepton protected the N-1 position while micrococcin rendered it more reactive. This difference correlates with the opposite effects of the two antibiotics on GTPase activity.

Anti-Bacterial Agents↗

PubMed1989

Archaebacterial DNA-dependent RNA polymerases testify to the evolution of the eukaryotic nuclear genome.

Genes for DNA-dependent RNA polymerase components B, A, and C from the archaebacterium Sulfolobus acidocaldarius and for components B", B', A, and C from the archaebacterium Halobacterium halobium were cloned and sequenced. They are organized in gene clusters in the order above, which corresponds to the order of the homologous rpoB and rpoC genes in the corresponding operon of the Escherichia coli genome. Derived amino acid sequences of archaebacterial components A and C were aligned with each other and with the sequences of corresponding (largest) subunits from the archaebacterium Methanobacterium thermoautotrophicum, with sequences of various eukaryotic nuclear RNA polymerases I, II, and III, and with the sequence of the beta' component from E. coli polymerase. The archaebacterial genes for component A are homologous to about the first two-thirds of genes for the eukaryotic component A and the eubacterial component beta', and the archaebacterial genes for component C are homologous to the last third of the genes for the eukaryotic component A and the eubacterial component beta'. Unrooted phylogenetic dendrograms derived from both distance matrix and parsimony analyses show the archaebacteria are a coherent group closely related to the eukaryotic nuclear RNA polymerase II and/or III lineages. The eukaryotic polymerase I lineage appears to arise separately from a bifurcation with the eubacterial beta' component lineage.

Amino Acid Sequence↗

PubMed1989

The phylogenetic relations of DNA-dependent RNA polymerases of archaebacteria, eukaryotes, and eubacteria.

Unrooted phylogenetic dendrograms were calculated by two independent methods, parsimony and distance matrix analysis, from an alignment of the derived amino acid sequences of the A and C subunits of the DNA-dependent RNA polymerases of the archaebacteria Sulfolobus acidocaldarius and Halobacterium halobium with 12 corresponding sequences including a further set of archaebacterial A+C subunits, eukaryotic nuclear RNA polymerases, pol I, pol II, and pol III, eubacterial beta' and chloroplast beta' and beta" subunits. They show the archaebacteria as a coherent group in close neighborhood of and sharing a bifurcation with eukaryotic pol II and (or) pol IIIA components. The most probable trees show pol IA branching off from the tree separately at a bifurcation with the eubacterial beta' lineage. The implications of these results, especially for understanding the possibly chimeric origin of the eukaryotic nuclear genome, are discussed.

Amino Acid Sequence↗

PubMed1989

Comparison of transfer RNA and ribosomal RNA intron splicing in the extreme thermophile and archaebacterium Desulfurococcus mobilis.

The structure of the exon-intron boundary was compared for an intron within 23S ribosomal RNA of Desulfurococcus mobilis and a newly discovered intron in tRNA(Met) from the same organism. The occurrence of a putative common structural feature suggests that intron excision occurs by the same mechanism. The possible recognition of this structural feature by the cleavage enzyme was investigated for the ribosomal RNA intron using RNA substrates exhibiting various exon and intron deletions. The results support the involvement of the structural features in the cleavage process. The evolutionary implications of these results are considered.

Archaea↗

PubMed1988

Domain VI of Escherichia coli 23 S ribosomal RNA. Structure, assembly and function.

Domain VI at the 3' end of the 23 S ribosomal RNA from Escherichia coli was prepared using the in vitro T7 RNA polymerase system. Its structure was examined by probing with ribonucleases and chemical reagents, including a psoralen derivative, of various nucleotide specificities, using a reverse transcriptase procedure for analysis. The data provided support for the most recent secondary structure derived from phylogenetic sequence comparisons and for additional structuring that was inferred from earlier experimental data. Moreover, the structure was essentially the same in the free domain, in renatured 23 S RNA and in 50 S subunits. Protein L3 bound to the isolated domain and its binding site was located at a long-range double helix containing a large internal loop. This structure is unusual for a protein-RNA binding site and it may characterize a new (third) class of site. Protein L3 has been implicated, together with L24, in initiating assembly of the 50 S subunit and it shares the exceptional property with L24 that it binds adjacent to the junction of two RNA domains from where it can maximally influence RNA folding. Protein L6 also assembled to domain VI and, in a control experiment, protein L2 bound to isolated domain IV. Domain VI was largely inaccessible in the 50 S subunit and the few accessible RNA sites occurred mainly within conserved sequence regions that constitute potential functional sites. alpha-Sarcin inactivates ribosomes by cutting at one of these sites in 50 S subunits; it also recognized the same site in the free 23 S RNA and in the free domain. Both the EF-Tu ternary complex, and the EF-G ternary complex stabilized by fusidic acid or by a non-hydrolyzable GTP derivative, inhibited alpha-sarcin attack while non-enzymatically bound tRNA did not, thus providing evidence, more direct than before, for the involvement of the RNA region in a common elongation factor binding site.

Autoradiography↗

PubMed1988

Novel splicing mechanism for the ribosomal RNA intron in the archaebacterium Desulfurococcus mobilis.

The intron of the 23S rRNA gene of D. mobilis is excised from the pre-23S RNA at specific sites in vivo and subsequently ligated to form a stable circular RNA, with a normal 5'-3' phosphodiester bond, containing the entire intron sequence; 95% of this RNA codes for a protein of 194 amino acids that can be expressed in E. coli. Crude cell extracts from D. mobilis also induce a two-step slicing reaction in vitro, producing the same circular intron RNA but a low yield of ligated exons. Cleavage depends on the RNA structure adjacent to the cleavage site and yields a 3'-terminal phosphate. Splicing is enhanced by GTP, but does not require divalent metal ions. The cleavage and exon-splicing reactions resemble those found for tRNA introns in eukaryotes and a possible structural rationale for this similarity is considered together with its possible implications for the origin of eukaryotic rRNA and tRNA introns.

Amino Acid Sequence↗

PubMed1988

The importance of highly conserved nucleotides in the binding region of chloramphenicol at the peptidyl transfer centre of Escherichia coli 23S ribosomal RNA.

The peptidyl transfer site has been localized at the centre of domain V of 23S-like ribosomal RNA (rRNA) primarily on the basis of a chloramphenicol binding site. The implicated region constitutes an unstructured circle in the current secondary structural model which contains several universally conserved nucleotides. With a view to investigate the function of this RNA region further, four of these conserved nucleotides, including one indirectly implicated in chloramphenicol binding, were selected for mutation in Escherichia coli 23S rRNA using oligonucleotide primers. Mutant RNAs were expressed in vivo on a plasmid-encoded rRNA (rrnB) operon and each one yielded dramatically altered phenotypes. Cells exhibiting A2060----C or A2450----C transversions were inviable and it was shown by inserting the mutated genes after a temperature-inducible promoter that the mutant RNAs were directly responsible. In addition, a G2502----A transition caused a decreased growth rate, probably due to a partial selection against mutant ribosome incorporation into polysomes, while an A2503----C transversion produced a decreased growth rate and conferred resistance to chloramphenicol. All of the mutant RNAs were incorporated into 50S subunits, but while the two lethal mutant RNAs were strongly selected against in 70S ribosomes, the plasmid-encoded A2503----C RNA was preferred over the chromosome-encoded RNA, contrary to current regulatory theories. The results establish the critical structural and functional importance of highly conserved nucleotides in the chloramphenicol binding region. A mechanistic model is also presented to explain the disruptive effect of chloramphenicol (and other antibiotics) on peptide bond formation at the ribosomal subunit interface.

Base Sequence↗

PubMed1987

Structure and accessibility of domain I of Escherichia coli 23 S RNA in free RNA, in the L24-RNA complex and in 50 S subunits. Implications for ribosomal assembly.

Domain I of 23 S RNA of Escherichia coli was probed in renatured RNA, in the protein L24-RNA complex and in 50 S subunits with ribonucleases specific for single- and double-stranded regions and with chemical reagents specific for guanosines (N-1 and N-2), adenosines (N-1, N-7 and N-6), cytidines (N-3) and uridines (N-3). Reactive sites were detected by a reverse transcriptase procedure. The results support most new features of the latest version of the Santa Cruz/Urbana model of the secondary structure, which is based on evidence from sequence comparison. Most double-helical segments were reactive to cobra venom ribonuclease to some degree; the exceptions were the five "long-range" helices that are probably compactly folded within the structure. The data provide evidence for the occurrence of A(syn) X G(anti) pairings in internal loops and at the ends of some helices; they also support the existence of extensive higher-order structuring, especially within the interhelical regions, and are compatible with two of three tertiary interactions in the free RNA that were predicted from comparative sequence studies. Protein L24 is the only primary binding protein that associates with domain I and it strongly protects two sites against ribonuclease and chemical activity. Site A has the properties of a classic protein binding site and we conclude from four lines of evidence that it is the primary attachment site. Site B is rich in highly conserved, unpaired adenosine residues and lies in a potentially critical region of the structure adjoining a group of long-range helices; we infer that L24 binding here is related to the important role of L24 in initiating ribosomal assembly; the existence of both sites is supported, independently, by genetic experiments. L24-induced enhanced reactivities were detected throughout the domain and are consistent with a general "tuning" of the RNA structure. The RNA domain in the 50 S subunits is almost completely resistant to ribonucleases and only a few sites, mainly interhelical, are accessible to chemical reagents. The appearance of several newly reactive nucleotides in the subunit RNA and the enhancement of some others suggest that some minor conformational changes occur on assembly. Nevertheless, the minimal secondary structure of the renatured RNA appears to be retained. We draw the general conclusion that domain I is a highly structured domain that is important for initiating assembly and for the subsequent organization of the ribosome.

Binding Sites↗

PubMed1987

Gene organization, transcription signals and processing of the single ribosomal RNA operon of the archaebacterium Thermoproteus tenax.

The single ribosomal RNA (rRNA) operon from the extreme thermophile and archaebacterium Thermoproteus tenax was sequenced. Sites of transcriptional initiation and termination were established and the processing sites on the primary transcript were mapped with nuclease S1. The operon contained genes coding for 16S and 23S RNAs but lacked those coding for tRNA and 5S RNA. Transcription initiates 175 bp upstream from the start of the 16S RNA gene (Wich et al., EMBO J. 6, 523-528, 1987) and terminates 49 bp downstream from the 23S RNA gene within a long pyrimidine sequence. An open reading frame downstream from the rRNA operon is transcribed. The sequences bordering both 16S and 23S RNA genes can form putative processing stems in the primary transcript that involve the whole of the 16S-23S RNA spacer. The stems contain irregular features that constitute processing signals and are conserved in other archaebacteria. The 16S RNA stem is cut prior to that of the 23S RNA and RNA maturation follows. An unusual 14 bp helix can form between the extremities of the transcript such that the whole transcript is highly structured and a fork-like structure is formed together with the processing stems. The 23S RNA sequence was aligned with other available 23S-like RNA sequences (Leffers et al., J. Mol. Biol. 195, in press): a putative secondary structure exhibiting archaebacterial-specific features was deduced using comparative sequence analyses. A rooted phylogenetic tree was also derived for the archaebacteria that confirms their division into three major subgroups.

Archaea↗

PubMed1987

Evolutionary relationships amongst archaebacteria. A comparative study of 23 S ribosomal RNAs of a sulphur-dependent extreme thermophile, an extreme halophile and a thermophilic methanogen.

The 23 S RNA genes representative of each of the main archaebacterial subkingdoms, Desulfurococcus mobilis an extreme thermophile, Halococcus morrhuae an extreme halophile and Methanobacterium thermoautotrophicum a thermophilic methanogen, were cloned and sequenced. The inferred RNA sequences were aligned with all the available 23 S-like RNAs of other archaebacteria, eubacteria/chloroplasts and the cytoplasm of eukaryotes. Universal secondary structural models containing six major structural domains were refined, and extended, using the sequence comparison approach. Much of the present structure was confirmed but six new helices were added, including one that also exists in the eukaryotic 5.8 S RNA, and extensions were made to several existing helices. The data throw doubt on whether the 5' and 3' ends of the 23 S RNA interact, since no stable helix can form in either the extreme thermophile or the methanogen RNA. A few secondary structural features, specific to the archaebacterial RNAs were identified; two of these were supported by a comparison of the archaebacterial RNA sequences, and experimentally, using chemical and ribonuclease probes. Seven tertiary structural interactions, common to all 23 S-like RNAs, were predicted within unpaired regions of the secondary structural model on the basis of co-variation of nucleotide pairs; two lie in the region of the 23 S RNA corresponding to 5.8 S RNA but they are not conserved in the latter. The flanking sequences of each of the RNAs could base-pair to form long RNA processing stems. They were not conserved in sequence but each exhibited a secondary structural feature that is common to all the archaebacterial stems for both 16 S and 23 S RNAs and constitutes a processing site. Kingdom-specific nucleotides have been identified that are associated with antibiotic binding sites at functional centres in 23 S-like RNAs: in the peptidyl transferase centre (erythromycin-domain V) the archaebacterial RNAs classify with the eukaryotic RNAs; at the elongation factor-dependent GTPase centre (thiostrepton-domain II) they fall with the eubacteria, and at the putative amino acyl tRNA site (alpha-sarcin-domain VI) they resemble eukaryotes. Two of the proposed tertiary interactions offer a structural explanation for how functional coupling of domains II and V occurs at the peptidyl transferase centre. Phylogenetic trees were constructed for the archaebacterial kingdom, and for the other two kingdoms, on the basis of the aligned 23 S-like RNA sequences.(ABSTRACT TRUNCATED AT 400 WORDS)

Archaea↗

PubMed1987

Novel expression of the ribosomal RNA genes in the extreme thermophile and archaebacterium Desulfurococcus mobilis.

The single 16S/23S rRNA operon of the sulfur-dependent extreme thermophile Desulfurococcus mobilis is transcriptionally uncoupled from the single 5S RNA gene. Transcription of the former operon initiates at five sites, P5-P1. RNA polymerases intiating at sites P5-P2 terminate and then reinitiate at the next downstream site, thus providing a queue of polymerases at the primary initiation site P1. Putative promoter sequences that are AT-rich lie upstream from each site. Termination occurs at two main sites within long pyrimidine sequences downstream from the 23S RNA gene. A processing site for both large RNAs occurs at a secondary structural feature common to other archaebacterial rRNAs. Transcription of 5S RNA starts mainly at P1/5S at the 5' end of the gene with a low level of initiation from two upstream sites; a similar mechanism of initiation-termination-reinitiation appears to operate and the sites are all preceded by AT-rich promoter-like sequences. Termination occurs a few nucleotides beyound the 3' end of the 5S RNA. The mature RNA contains 133 nucleotides, has a 78% GC bp content and exhibits an unusually extended version of helix I with heterogeneous ends. The expression of the rRNA genes shows characteristics of eukaryotes, eubacteria and archaebacteria.

Journal Article↗

PubMed1987

Xenopus transcription factor IIIA binds primarily at junctions between double helical stems and internal loops in oocyte 5S RNA.

RNases and chemical probes were used to study the accessibility of each nucleotide of 5S RNA in the native and reconstituted 7S particle from Xenopus laevis oocytes. RNase or chemically treated 5S RNA from intact 7S particles was isolated and analysed using an oligodeoxynucleotide primer and reverse transcriptase. The results were superimposed on a cylindrical projection of an RNA double helix and the protection effects were shown to cluster at two regions on the molecular surface. A three-dimensional model is proposed for the 7S particle in which protein-RNA contacts occur mainly in the major groove of 5S RNA.

Animals↗

PubMed1987

A plasmid-coded and site-directed mutation in Escherichia coli 23S RNA that confers resistance to erythromycin: implications for the mechanism of action of erythromycin.

Primer-directed mutagenesis was employed to introduce an A2058----G transition in plasmid-encoded Escherichia coli 23S RNA at a site that has been implicated, indirectly, in erythromycin binding. The mutation raises the growth tolerance of cells from 30 to 300 micrograms/ml of erythromycin, and cells grown in the presence of erythromycin contain ribosomes with high levels of mutated 23S RNA. In these cells, wild type 50S subunits 'fall off' the message and are selectively degraded, possibly as a result of an erythromycin-induced conformational change. A fast in vitro poly(U) assay revealed minimal effects of erythromycin on elongation beyond tetrapeptides. We correlated these results with the literature data and concluded that erythromycin acts immediately post-initiation and directly, or indirectly, destabilizes mRNA-bound 70S ribosomes, and prevents their recycling by causing 50S subunit degradation.

Autoradiography↗

PubMed1986

Congenital posterior urethral polyps.

Congenital posterior urethral polyps are benign growths that can cause a variety of symptoms in young boys. They are pathologically distinct growths usually discovered by voiding cystourethrography. We present 5 cases of congenital posterior urethral polyps, and discuss the presentation and pathophysiological status of these lesions. These lesions must be considered in the differential diagnosis of voiding dysfunction in young boys.

Child↗

PubMed1985

Alternative conformers of 5S ribosomal RNA and their biological relevance.

Different conformational states of Escherichia coli 5S ribosomal RNA that may participate in protein biosynthesis have been either detected experimentally or predicted on the basis of phylogenetic sequence comparisons. The A conformer exists in a high-salt form (AH) that binds ribosomal proteins and assembles into the 50S subunit and in a low-salt form (AL), of uncertain biological relevance, that binds at least one ribosomal protein and differs in tertiary structure from the AH form. Experimentally, the AH form has been investigated comprehensively and the AL form partially. There is also a B conformer that exhibits an altered secondary structure and does not assemble with ribosomal proteins. For this conformer exhibits an altered secondary structure and does not assemble with ribosomal proteins. For this conformer to be functionally active, it must be both discrete and universal among 5S RNAs. Here, we examine its structure by employing single and double strand specific ribonucleases and nucleotide-specific chemical reagents. We demonstrate that the B form exhibits a secondary structure only a part of which is both universal and conformationally homogeneous, and we conclude, therefore, that the whole B form cannot participate in protein biosynthesis. We note, however, that progressive structural changes occur during the transitions AH----AL----B and provide evidence that the structural alteration during the transition AH----AL may be universal, which reinforces the view that the AL form is of biological relevance.

Electrophoresis, Polyacrylamide Gel↗

PubMed1985

Comparison of eubacterial and eukaryotic 5S RNA structures: a chemical modification study.

The 5S RNAs from Bacillus stearothermophilus and Saccharomyces cerevisiae were probed by nucleotide-specific reagents, with a view to compare and contrast their higher order structures. The progressive unfolding of the RNAs during heating, in the presence and absence of magnesium, was monitored. Evidence was provided for the double-helical segments which occur in the secondary structural models of both RNAs. The results also placed constraints on the possible structuring of the remainder of the RNA and yielded some insight into ways of folding up the molecule. Together with the data from our earlier studies, employing ribonucleases, these results provide a detailed picture of the structuring and topography of the 5S RNAs. The main structural differences between the eubacterial and eukaryotic RNAs occur throughout the loop D/helix IV/loop E/helix V arm; in particular strong evidence is provided for loop D of the eukaryotic RNA being involved in a tertiary interaction.

Geobacillus stearothermophilus↗