Biomedical subjects

R A Garrett

Publications and source records attributed to R A Garrett.

At least 55 records · Page 3Linked to original sources

PubMed1995

Role for the highly conserved region of domain IV of 23S-like rRNA in subunit-subunit interactions at the peptidyl transferase centre.

The function of the highly conserved and accessible region of domain IV of 23S rRNA (positions 1900-1981 in Escherichia coli 23S rRNA) was investigated by subjecting it to a random mutagenesis procedure that produced single-site mutations efficiently. Nine single-site mutants were selected that were recessive lethal. High levels of mutated 23S rRNA were expressed in E. coli and extracted ribosomes were investigated for their content of mutated rRNA. The peptidyl transferase activity of the ribosomes was also estimated using a newly developed method involving selective inhibition of chromosome-encoded ribosomes by clindamycin. Two of the mutants, U1940A and U1955G, yielded 50S subunits that were defective in subunit-subunit association but active in peptidyl transferase activity and five, U1926C, U1946C, U1979C, U1982A and G1984A, produced 50S subunits that were defective in both subunit-subunit interactions and peptidyl transferase activity. We infer that the large conserved rRNA region generates a complex structure that plays an essential role in maintaining and modulating subunit-subunit interactions and argue that its involvement in the peptidyl transferase centre is secondary, possibly involving the correct alignment of protein L2.

Base Sequence↗

PubMed1995

Fine structure of the peptidyl transferase centre on 23 S-like rRNAs deduced from chemical probing of antibiotic-ribosome complexes.

Ribosomal binding sites were investigated for the diverse group of antibiotics: anisomycin, anthelmycin, blasticidin S, bruceantin, carbomycin, chloramphenicol, griseoviridin, narciclasine, T2 toxin, tylosin and virginiamycin M1 all of which are considered to inhibit the peptidyl transferase reaction by different mechanisms. The drugs also exhibit differing degrees of specificity for bacterial, archaeal and eukaryotic ribosomes despite a high level of conservation of sequence and secondary structure at the peptidyl transferase centre of the 23 S-like rRNAs. The drug binding sites were characterized by incubating each antibiotic with ribosomes from a bacterium, an archaeon and a eukaryote and chemically probing the 23 S-like rRNA. The complexity of the changes in reactivity ranged from one or two nucleotides (anthelmycin, narciclasine) to eight or nine (virginiamycin M1) and it was inferred, at least for those drugs producing complex changes, that they induce, and stabilize, a particular functional conformer in the peptidyl transferase centre. The results were correlated with literature data on both ribosomal ligand binding and the putative inhibitory mechanisms of the drugs, and the following inferences are made concerning the fine structure of the peptidyl transferase centre. (1) An irregular secondary structural motif, which includes unpaired A2439 (Escherichia coli numbering), lies close to the catalytic centre; (2) nucleotides A2451 and C2452 contribute to a site for the binding of the side chains of aromatic amino acids; (3) the P-substrate site encompasses U2585, U2506 and, possibly, a site in domain IV (A1787), and (4) the sequence A2058 to A2062 and nucleotide U2609 contribute to, or modulate, the start of the peptide channel. No drug effects were found that could be directly attributed to an A-site and the possibility is raised that, if it exists, it consists mainly of ribosomal proteins. However, two drugs T2 toxin and virginiamycin M1 protected the only nucleotide in the peptidyl transferase loop region (C2394) associated with the E-site. Finally, it is proposed that the putative sub-sites are physically separated, that some drugs bind to more than one of them, and that they are conformationally interdependent.

Anti-Bacterial Agents↗

PubMed1994

Cross-hypersensitivity effects of mutations in 23 S rRNA yield insight into aminoacyl-tRNA binding.

Halobacterium halobium cells contain one set of rRNA genes per genome. They have been used to characterize spontaneous mutants, carrying single nucleotide mutations in their rRNAs, that are resistant to different ribosomal antibiotics. Here we demonstrate that two different mutants that show resistance to thiostrepton, an inhibitor of the ribosomal GTPase-centre, are hypersensitive to amicetin, and other antibiotics, which act at the peptidyl transferase centre. Conversely, an amicetin-resistant mutant exhibits hypersensitivity to thiostrepton. A model is presented in which the two mutated sites, which are widely separated in the primary and secondary structure of 23 S rRNA, both participate in A-site binding of aminoacyl-tRNA.

Anti-Bacterial Agents↗

PubMed1994

Purification and characterization of two forms of I-DmoI, a thermophilic site-specific endonuclease encoded by an archaeal intron.

The archaeal intron in the 23 S rRNA gene of the hyperthermophile Desulfurococcus mobilis has previously been shown to encode a site-specific DNA endonuclease that contains the LAGLIDADG motif. The enzyme, I-DmoI, has been shown to be active in two forms when expressed in vitro, from RNAs representing either the linear (I-DmoIl) or circular (I-DmoIc) intron. In this study we have overexpressed I-DmoIl and I-DmoIc and purified the enzymes from Escherichia coli. The optimal conditions for the enzymatic activity in vitro were determined, and the enzyme was used to delimit the recognition boundary on its DNA substrate (14-20 nucleotides), an intronless 23 S rRNA gene. Despite belonging to the archaeal kingdom, and being the product of a hyperthermophile, I-DmoI shares many properties with LAGLIDADG intron and intein endonucleases in other kingdoms. These results support the view that these phylogenetically diverse enzymes, which function to mobilize the DNA sequences that encode them, share a common ancestry.

Amino Acid Sequence↗

PubMed1994

Structural characteristics of the stable RNA introns of archaeal hyperthermophiles and their splicing junctions.

Five pre-23 S rRNA introns have been characterized for hyperthermophilic archaea. The small ones constitute stable core structures while the three larger ones contain, in addition, open reading frames, two of which encode homing-type endonucleases. The higher order structures of in vitro transcripts of the introns and their exon-intron junctions were examined using chemical and ribonuclease probes specific for unpaired nucleotides, double helical regions and helix-loop junctions. The experimental data support both the formation of a "bulge-helix-bulge" structural motif at the exon-intron junctions, which is recognized by a cleavage enzyme, and the presence of an adjacent core structure consisting of a long, stable, stem-loop structure. Moreover, the data were used to derive secondary structural models for the three large introns which form stable, circular, RNA species, in vivo. Each exhibits extensive secondary structure and additional, as yet undefined, higher order structure, compatible with their high level of stability in vivo; limited structural similarities were detected between the two introns encoding the homing-type endonucleases. The data are compatible with the hypothesis that RNA introns can be transmitted between archaeal hyperthermophile cells.

Archaea↗

PubMed1994

DNA substrate specificity and cleavage kinetics of an archaeal homing-type endonuclease from Pyrobaculum organotrophum.

The protein encoded by intron 1 of the single 23S rRNA gene of the archaeal hyperthermophile Pyrobaculum organotrophum was isolated and shown to constitute a homing-type DNA endonuclease, I-PorI. It cleaves the intron- 23S rDNA of the closely related organism Pyrobaculum islandicum near the site of intron insertion in Pb.organotrophum. In contrast, no endonuclease activity was detected for the protein product of intron 2 of the same gene of Pb.organotrophum which, like I-PorI, carries the LAGLI-DADG motif, common to group I intron-encoded homing enzymes. I-PorI cleaves optimally at 80 degrees C, with kcat and Km values of about 2 min-1 and 4 nM, respectively, and generates four nucleotide 3'-overhangs and 5'-phosphates. It can cleave a 25 base pair DNA fragment encompassing the intron insertion site. A mutation-selection study established the base pair specificity of the endonuclease within a 17 bp region, from positions -6 to +11 with respect to the intron-insertion site. Four of the essential base pairs encode the sequence involved in the intron-exon interaction in the pre-rRNA that is required for recognition by the RNA splicing enzymes. Properties of the enzyme are compared and contrasted with those of eucaryotic homing endonucleases.

Amino Acid Sequence↗

PubMed1994

A conserved secondary structural motif in 23S rRNA defines the site of interaction of amicetin, a universal inhibitor of peptide bond formation.

The binding site and probable site of action have been determined for the universal antibiotic amicetin which inhibits peptide bond formation. Evidence from in vivo mutants, site-directed mutations and chemical footprinting all implicate a highly conserved motif in the secondary structure of the 23S-like rRNA close to the central circle of domain V. We infer that this motif lies at, or close to, the catalytic site in the peptidyl transfer centre. The binding site of amicetin is the first of a group of functionally related hexose-cytosine inhibitors to be localized on the ribosome.

Anti-Bacterial Agents↗

PubMed1994

A spontaneous point mutation in the single 23S rRNA gene of the thermophilic arachaeon Sulfolobus acidocaldarius confers multiple drug resistance.

Development of transformable vectors for thermophilic archaea requires the characterization of appropriate selectable marker genes. Many antibiotic inhibitors of protein biosynthesis are known to bind to rRNA; therefore, we screened 14 for their capacity to inhibit growth of the thermophilic archaeon Sulfolobus acidocaldarius. Carbomycin, celesticetin, chloramphenicol, puromycin, sparsomycin, tetracycline, and thiostrepton all inhibited growth by different degrees. Spontaneous drug-resistant mutants were isolated from plates containing celesticetin or chloramphenicol. Six mutants from each plate exhibited a C-2585-to-U transition in the peptidyl transferase loop of 23S rRNA (corresponding to C-2452 in Escherichia coli 23S rRNA). The single-site mutation also conferred resistance to carbomycin. The mutated 23S rRNA gene provides a potentially useful and dominant marker for a thermophilic archael vector.

Base Sequence↗

PubMed1993

A site-specific endonuclease encoded by a typical archaeal intron.

The protein encoded by the archaeal intron in the 23S rRNA gene of the hyperthermophile Desulfurococcus mobilis is a double-strand DNase that, like group I intron homing endonucleases, is capable of cleaving an intronless allele of the gene. This enzyme, I-Dmo I, is unusual among the intron endonucleases in that it is thermostable and is expressed only from linear and cyclized intron species and not from the precursor RNA. However, in analogy to its eukaryotic counterparts, but unlike the bacteriophage enzymes, I-Dmo I makes a staggered double-strand cut that generates 4-nt 3' extensions. Additionally, although the archaeal and group I introns have entirely different structural properties and splicing pathways, I-Dmo I shares sequence similarity, in the form of the LAGLI-DADG motif, with group I intron endonucleases of eukaryotes. These observations support the independent evolutionary origin of endonucleases and intron core elements and are consistent with the invasive potential of endonuclease genes.

Amino Acid Sequence↗

PubMed1992

Protein-coding introns from the 23S rRNA-encoding gene form stable circles in the hyperthermophilic archaeon Pyrobaculum organotrophum.

Two archaeal introns have been discovered in the single-copy 23S rRNA-encoding gene of the hyperthermophile, Pyrobaculum organotrophum. After excision from rRNA transcripts, both introns circularize and are stably retained in the cell. Putative proteins encoded by the introns and covering most of the intron sequence share a decapeptide motif with proteins encoded by another archaeal intron and by group I introns.

Amino Acid Sequence↗

PubMed1992

Introducing mutations into the single-copy chromosomal 23S rRNA gene of the archaeon Halobacterium halobium by using an rRNA operon-based transformation system.

A vector-transformation system is described that permits replacement of a portion of the single rRNA operon of the archaeon Halobacterium halobium with a homologous fragment from a vector-borne gene. The vector construct contains three functional sections: (i) an entire H. halobium rRNA operon with two selective mutations in the 23S rRNA gene, the substitutions of A----G at position 1159 conferring resistance to thiostrepton and C----U at position 2471 conferring resistance to anisomycin; (ii) the complete pHSB1 plasmid from Halobacterium sp. SB3, which interferes with vector maintenance in the transformed halobacterial cells; and (iii) a segment of the pBR322 plasmid that permits vector replication in Escherichia coli. Transformation of H. halobium with the vector plasmid generates cells resistant to both anisomycin and thiostrepton that can be selected for, and discriminated from spontaneous mutants, by a two-step selection procedure. After transformation, the plasmid recombines homologously with the chromosome so that the plasmid-borne rDNA segment with resistance markers substitutes for the corresponding region of the chromosomal rRNA operon, and the transforming plasmid is lost. Eventually, this leads to a homogeneous population of the mutant ribosomes in the cell. Other mutations that are engineered in the vector-borne rRNA sequences can be transferred to the chromosomal rRNA operon concomitantly with the selective markers. The system has considerable potential for ribosomal engineering.

Anisomycin↗

PubMed1991

Attachment sites of primary binding proteins L1, L2 and L23 on 23 S ribosomal RNA of Escherichia coli.

The attachment sites of the primary binding proteins L1, L2 and L23 on 23 S ribosomal RNA of Escherichia coli were examined by a chemical and ribonuclease footprinting method using several probes with different specificities. The results show that the sites are confined to localized RNA regions within the large ribonuclease-protected ribonucleoprotein fragments that were characterized earlier. They are as follows: (1) L1 recognizes a tertiary structural motif in domain V centred on two interacting internal loops; the main protein interaction sites occur at the internal loop/helix junctions. (2) The L2 site constitutes a single irregular stem/loop structure in the centre of domain IV where non-Watson-Crick pairing is likely to occur. (3) L23 recognizes a tertiary structural motif involving a single terminal loop structure and part of an adjacent internal loop at the centre of domain III. Each of the three primary binding proteins, whose presence is essential for ribosomal assembly, has been associated with important ribosomal functions: L1 lies in the E-site for deacylated tRNA binding while L2 and L23 have been implicated in the P and A substrate sites, respectively, of the peptidyl transferase centre. Moreover, each of the protein sites, but particularly those of L2 and L23, lies at the centre of RNA domains where they can maximally influence both the assembly of secondary binding proteins and the function of the RNA region.

Base Sequence↗

PubMed1991

Ribosomal RNA introns in archaea and evidence for RNA conformational changes associated with splicing.

The single 23S rRNA gene of the archaeon Staphylothermus marinus exhibits two introns which, at the RNA level, are located in highly conserved regions of domains IV and V. The RNA introns, which are 56 and 54 nucleotides long, respectively, can form single hairpin structures. In vivo, RNA splicing occurs efficiently, whereas in vitro pre-rRNA transcripts containing each intron were cleaved efficiently when incubated with archaeal cell extracts but were poorly ligated. The introns are cleaved by a mechanism which differs from the mechanisms of eukaryotic rRNA introns but resembles those of the rRNA intron of Desulfurococcus mobilis and the archaeal tRNA introns. The cleavage enzyme recognizes and cuts a putative bulge-helix-bulge structure that can form at the archaeal exon-intron junctions. Using a phylogenetic sequence comparison approach, we define the parts of this structural feature that are essential for cleavage. We also provide evidence for conformational changes occurring in the S. marinus 23S RNA, after cleavage, at both exon-exon junctions, which may account for the low yields of ligation observed in vitro.

Archaea↗

PubMed1991

Archaeal rRNA operons.

Ribosomal RNA (rRNA) operons of the archaea reflect both the unity and the diversity of this third primary taxon. They have proven to be a rich source of both molecular biological and phylogenetic information.

Archaea↗

PubMed1991

Chloramphenicol resistance mutations in the single 23S rRNA gene of the archaeon Halobacterium halobium.

A broad range of antibiotics affecting protein biosynthesis were screened for their ability to inhibit growth of the archaeon Halobacterium halobium. Only a few drugs, including chloramphenicol, produced inhibitory effects. Mutants which showed increased resistance to chloramphenicol were isolated; of the nine tested, eight exhibited a C----U transition at position 2471 and the ninth had an A----C transversion at position 2088 of 23S rRNA. A double mutant containing both C----U (position 2471) and A----C (position 2088) mutations was isolated, but the level of its chloramphenicol resistance did not exceed that of either single-point mutant. Inferences are made concerning the functional significance of the conserved nucleotides in rRNAs.

Base Sequence↗

PubMed1990

Characterization of the binding sites of protein L11 and the L10.(L12)4 pentameric complex in the GTPase domain of 23 S ribosomal RNA from Escherichia coli.

Ribonuclease and chemical probes were used to investigate the binding sites of ribosomal protein L11 and the pentameric complex L10.(L12)4 on Escherichia coli 23 S RNA. Protein complexes were formed with an RNA fragment constituting most of domains I and II or with 23 S RNA and they were investigated by an end-labelling method and a reverse transcriptase procedure, respectively. The results demonstrate that the two protein moieties bind at adjacent sites within a small RNA region. The L11 binding region overlaps with those of the modified peptide antibiotics thiostrepton and micrococcin and is constrained structurally by a three-helix junction while the L10.(L12)4 site is centred on an adjacent internal loop. The secondary structure of the whole region was determined in detail by the phylogenetic sequence comparison method, and the results for the L11 binding region, together with the experimental data, were used in a computer graphics approach to build a partial RNA tertiary structural model. The model provides insight into the topography of the L11 binding site. It also provides a structural rationale for the mutually co-operative binding of protein L11 with the antibiotics thiostrepton and micrococcin, and with the L10.(L12)4 protein complex.

Base Sequence↗

PubMed1990

Secondary structural elements exclusive to the sequences flanking ribosomal RNAs lend support to the monophyletic nature of the archaebacteria.

Several sequences flanking the large rRNA genes of several transcripts from extreme thermophiles, extreme halophiles, and methanogens were aligned and analyzed for the presence of common primary and secondary structural features, which would bear on the concept of monophyletic archaebacteria. Few sequences were common to all the archaebacterial transcripts, and these were confined to short regions generally flanking putative double helices. At a secondary structural level, however, in addition to the previously characterized processing stems of the 16S and 23S RNAs, four helices were detected that were common to the archaebacterial transcripts: two in the 16S RNA leader sequence and two in the 16S-23S RNA spacer. Although all of these helices vary in size and form from organism to organism, three of them contain double helical segments that are strongly supported by compensating base changes among the three archaebacterial groups. Three extreme halophiles exhibited two additional helices in their relatively large spacers and a further helix preceding the 5S RNA, which are also supported by compensating base changes. Ribosomal RNA transcripts from eubacteria/chloroplasts and eukaryotes were also examined for secondary structural features with locations and forms corresponding to those of the archaebacteria, but none were detected. The analysis provides support for the monophyletic nature of the archaebacteria and reinforces their differences from eubacteria/chloroplasts and eukaryotes.

Archaea↗

PubMed1989

A unique tRNA intron in the variable loop of the extreme thermophile Thermofilum pendens and its possible evolutionary implications.

We describe, for the first time, an intron that is located in the variable loop of a tRNA. It is 18 nucleotides long and occurs within a precursor tRNAGly from the extreme thermophile Thermofilum pendens. The variable loop is less critical for tRNA function than the anticodon arm where other class III introns occur. This raises the possibility that tRNAs with a large variable loop (type II) arose from those with a small variable loop (type I) by retaining a splicing-deficient intron.

Base Sequence↗