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PubMed1989

Sequence and secondary structure of the central domain of Drosophila 26S rRNA: a universal model for the central domain of the large rRNA containing the region in which the central break may happen.

An 890-bp sequence from the central region of Drosophila melanogaster 26S ribosomal DNA (rDNA) has been determined and used in an extensive comparative analysis of the central domain of the large subunit ribosomal RNA (lrRNA) from prokaryotes, organelles, and eukaryotes. An alignment of these different sequences has allowed us to precisely map the regions of the central domain that have highly diverged during evolution. Using this sequence comparison, we have derived a secondary structure model of the central domain of Drosophila 26S ribosomal RNA (rRNA). We show that a large part of this model can be applied to the central domain of lrRNA from prokaryotes, eukaryotes, and organelles, therefore defining a universal common structural core. Likewise, a comparative study of the secondary structure of the divergent regions has been performed in several organisms. The results show that, despite a nearly complete divergence in their length and sequence, a common structural core is also present in divergent regions. In some organisms, one or two of the divergent regions of the central domain are removed by processing events. The sequence and structure of these regions (fragmentation spacers) have been compared to those of the corresponding divergent regions that remain part of the mature rRNA in other species.

Animals↗

PubMed2000

Structural variation in the 16S-23S rRNA intergenic spacers of Vibrio parahaemolyticus.

The 16S-23S rRNA intergenic spacers (IGS) of Vibrio parahaemolyticus were PCR-amplified and cloned with pT7Blue T vector. A total of six clones were isolated dependent on size difference. The clones were different with respect to both the number and the composition of the tRNA genes included, and were designated IGS-0, IGS-E, IGS-IA, IGS-AE, IGS-EKV and IGS-EKAV. IGS-EKAV included the cluster of tRNA(Glu)-tRNA(Lys)-tRNA(Ala)-tRNA(Val); IGS-EKV, tRNA(Glu)-tRNA(Lys)-tRNA(Val); IGS-AE, tRNA(Ala)-tRNA(Glu); IGS-IA, tRNA(Ile)-tRNA(Ala); and IGS-E, tRNA(Glu). IGS-0 had no tRNA gene. Some similarities were found in the nucleotide sequence of the non-coding regions flanked by the tRNA genes. The structure difference found in the spacers is meaningful for elucidating the evolutionary line of each ribosomal RNA operon and the profile is applicable as a molecular marker of the bacterium.

Cloning, Molecular↗

PubMed1985

Quantitative structural analysis of eukaryotic ribosomal RNA by scanning transmission electron microscopy.

The conformation of 28S ribosomal RNA isolated from baby hamster kidney cells was studied by scanning transmission electron microscopy (STEM) and circular dichroic spectroscopy to establish the conditions under which STEM images of unstained freeze-dried rRNA are a meaningful representation of the conformation of rRNA in solution. We have determined the conformation of 28S rRNA under various buffer conditions, the molecular mass, the mass distribution, and the number of polynucleotide strands within the individual molecules, and the apparent radii of gyration. The 28S rRNA molecule is highly extended in water and becomes compact with increasing ionic strength. However, even in the "reconstitution buffer" (30 mM Tris/HCl/20 mM MgCl2/360 mM KCl, pH 7.6) the compactness does not reach a state in which the rRNA molecule appears structurally similar to the 60S ribosomal subunit. Our approach has a broad application in high-resolution structural studies of nucleic acids and nucleic acid-protein interactions.

Animals↗

PubMed1983

Location of 5.8 S rRNA contact sites in 28 S rRNA and the effect of alpha-sarcin on the association of 5.8 S rRNA with 28 S rRNA.

We have constructed phage M13mp7 clones each containing the coding strand from one of three restriction fragments which collectively span the mouse 28 S rRNA gene with the exception of the 3'-terminal approximately 500 base pairs. When hybridized to 28 S rRNA, only the fragment containing the 5'-terminal 1400 nucleotides of the gene inhibited the annealing of 5.8 S rRNA to the 28 S rRNA. The same results were obtained when either the 5'- or 3'-terminal fragment of 5.8 S rRNA was used in lieu of intact 5.8 S rRNA, each of which had been shown to contain an independent 28 S rRNA contact site. However, alpha-sarcin, a cytotoxin that inhibits protein synthesis by hydrolyzing a phosphodiester bond near the 3' end of 28 S rRNA, produces a 3'-terminal 488-nucleotide fragment which exhibits a marginal capacity to anneal to 5.8 S rRNA. These results indicate that 5.8 S rRNA interacts predominantly with a structural domain near the 5' end of 28 S rRNA. This conclusion is consistent with base-pairing interactions between 5.8 S rRNA and 28 S rRNA based on the proposed secondary structures for Escherichia coli 23 S and yeast 26 S rRNAs. However, alpha-sarcin treatment of ribosomes affects the stability of the binding of 5.8 S rRNA to the 28 S rRNA, even though the toxin hydrolyzes a phosphodiester bond several thousand nucleotides from the proposed contact regions. Finally, mouse 5.8 S rRNA was shown to lack two internal nucleotides reported to be present in rat 5.8 S rRNA.

Animals↗

PubMed2002

Complete sequence and structure of ribosomal RNA gene of Heterosporis anguillarum.

The ribosomal RNA (rRNA) gene region of the microsporidium Heterosporis anguillarum has been examined. Complete DNA sequence data (4060 bp, GenBank Accession No. AF402839) of the rRNA gene of H. anguillarum are presented for the small subunit gene (SSU rRNA: 1359 bp), the internal transcribed spacer (ITS: 37 bp), and the large subunit gene (LSU rRNA: 2664 bp). The secondary structures of the H. anguillarum SSU and LSU rRNA genes are constructed and described. This is the first complete sequence of an rRNA gene published for a fish-infecting microsporidian species. In the phylogenetic analysis, the sequences, including partial SSU rRNA, ITS, and partial LSU rRNA sequences of the fish-infecting microsporidia, were aligned and analysed. The taxonomic position of H. anguillarum as suggested by Lom et al. (2000; Dis Aquat Org 43:225-231) is confirmed in this paper.

Anguilla↗

PubMed1991

Cloning, characterization and taxonomic significance of genes for the 5S ribosomal RNA of Leptonema illini strain 3055.

The genes encoding the 5S ribosomal RNA (rRNA) for Leptonema illini strain 3055 were isolated and sequenced. The 5S RNA molecule encoded was 117 nucleotides long. The genome of strain 3055 contained two genes for 5S rRNA that were located close together. The nucleotide sequences of the Leptonema illini genes exhibited less similarity to the rRNA gene of Leptospira interrogans strain Moulton and also to those of typical eubacterial genes than did the rRNA genes of other leptospires. However, the overall secondary structure of the 5S rRNA encoded exhibited a strong similarity to that of typical eubacterial 5S rRNA. Southern hybridization of the 5S rRNA gene probe with the genomic DNA of strain 965, which is currently classified as Leptospira biflexa, showed the latter to have close similarity to that of strain 3055. The physical map of strain 965 was quite similar to that of strain 3055 and was greatly different from that of any other strains of L. biflexa. In the organization of 5S rRNA genes, strain 965 is sufficiently different from other members of the genus Leptospira to be regarded as a member of the genus Leptonema.

Base Sequence↗

PubMed1980

Electron microscopic mapping of secondary structures in bacterial 16S and 23S ribosomal ribonucleic acid and 30S precursor ribosomal ribonucleic acid.

Electron microscopy revealed reproducible secondary structure patterns within partially denatured 16S and 23S ribosomal ribonucleic acid (rRNA) from Escherichia coli. When prepared with 50% formamide-100 mM ammonium acetate, 16S rRNA included two small hairpins that appeared in over 50% of all molecules. Three open loops were observed with frequencies of less than 25%. In contrast, 23S rRNA included a terminal open loop and two additional large structures in over 75% of all molecules. These secondary structure patterns were conserved in the 16S and 23S rRNA from Pseudomonas aeruginosa. The secondary structure of the 30S precursor rRNA from the ribonclease III-deficient E. coli mutant AB105 was mapped after partial denaturation in 70% formamide-100 mM ammonium acetate. Two large open loops were superimposed on the 16S and 23S rRNA secondary structure patterns. These loops were the most frequent structures found on the precursor, and their stems coincided with ribonuclease III cleavage sites. A tentative 5'-3 orientation was determined for the secondary structure patterns of 16S and 23S rRNA from their relative locations within 30S precursor rRNA. The relation of secondary structure to ribosomal protein binding and ribonuclease III cleavage is discussed.

Escherichia coli↗

PubMed1998

Empirical estimation of the reliability of ribosomal RNA alignments.

MOTIVATION: The automatic alignment of rRNA sequences can reproduce manual expert alignments with high, but not perfect, fidelity. We examine the use of empirical methods for the identification of regions of an alignment of a new sequence with an existing large alignment which can confidently be predicted to be correctly aligned. RESULTS: We show how to use a simple jack-knife procedure to derive an estimate of the reliability that is to be expected at each position of a large alignment of eukaryotic rRNA sequences. These reliabilities are then improved using measures that are specific to the input sequence. Regions where the sequence-specific reliability method performs particularly well are identified and seen to correspond with elements in the structure of the rRNA molecules that vary between species in the alignment. We also compare these reliability measures to an algorithmic alignment stability measure. AVAILABILITY: The software is available free of charge by sending an e-mail message to emmet@chah.ucc.ie. CONTACT: emmet@chah.ucc.ie

Algorithms↗

PubMed2006

High resolution mapping of ribosomal DNA in early mouse embryos by fluorescence in situ hybridization.

The nucleolar precursor bodies (NPBs) are numerous discrete entities present in the nuclei of early mammalian embryos, which structurally support active rRNA genes. However, whether all rRNA genes, including those not transcribed, are spatially associated with NPBs, and moreover what is the general arrangement of ribosomal DNA (rDNA) in early mouse embryos, still remain unanswered questions. In our study, we examined the localization of rDNA in transcriptionally silent (one-cell and early two-cell) and transcriptionally active (late two-cell) mouse embryos by highly sensitive fluorescence in situ hybridization with probes complementary to mouse rDNA repeats. The results obtained showed that irrespective of the rDNA transcriptional status, one or more NPBs per nucleus were not structurally associated with rDNA. These observations support the idea that NPBs are heterogeneous in their ability to recruit rRNA genes and thus to participate in reassembly of the mature nucleolus. As in somatic cells, and despite the absence of the characteristic nucleoli, the general arrangement of rRNA genes in early mouse embryos reflected the intensity of rDNA transcription. Ribosomal RNA genes were unequally distributed with respect to repeat putative copy numbers between nucleolar organizing region (NOR)-bearing chromosomes at the first cleavage division, and more strikingly, between sister chromatid NORs of a single nucleolar organizing chromosome. The latter indicates that sister chromatids might harbor various numbers of rRNA gene copies, and that the genes might be unequally distributed between the two blastomeres during the first cleavage mitosis.

Animals↗

PubMed2001

Proposed secondary structure of eukaryote specific expansion segment 15 in 28S rRNA from mice, rats, and rabbits.

The expansion segments in eukaryotic ribosomal RNAs are additional RNA sequences not found in the RNA core common to both prokaryotes and eukaryotes. These regions show large species-dependent variations in sequence and size. This makes it difficult to create secondary structure models for the expansion segments exclusively based on phylogenetic sequence comparison. Here we have used a combination of experimental data and computational methods to generate secondary structure models for expansion segment 15 in 28S rRNA in mice, rats, and rabbits. The experimental data were collected using the structure sensitive reagents DMS, CMCT, kethoxal, micrococcal nuclease, RNase T(1), RNase CL3, RNase V(1), and lead(II) acetate. ES15 was folded with the computer program RNAStructure 3.5 using modification data and phylogenetic similarities between different ES15 sequences. This program uses energy minimization to find the most stable secondary structure of an RNA sequence. The presented secondary structure models include several common structural motifs, but they also have characteristics unique to each organism. Overall, the secondary structure models showed indications of an energetically stable but dynamic structure, easily accessible from the solution by the modification reagents, suggesting that the expansion segment is located on the ribosomal surface.

Aldehydes↗

PubMed2006

Molecular organization of 5S rDNAs in Rajidae (Chondrichthyes): Structural features and evolution of piscine 5S rRNA genes and nontranscribed intergenic spacers.

The genomic and gene organisation of 5S rDNA clusters have been extensively characterized in bony fish and eukaryotes, providing general issues for understanding the molecular evolution of this multigene DNA family. By contrast, the 5S rDNA features have been rarely investigated in cartilaginous fish (only three species). Here, we provide evidence for a dual 5S rDNA gene system in the Rajidae by sequence analysis of the coding region (5S) and adjacent nontranscribed spacer (NTS) in five Mediterranean species of rays (Rajidae), and in a large number of piscine taxa including lampreys and bony fish. As documented in several bony fish, two functional 5S rDNA types were found here also in the rajid genome: a short one (I) and a long one (II), distinguished by distinct 5S and NTS sequences. That the ancestral piscine genome had these two 5S rDNA loci might be argued from the occurrence of homologous dual gene systems that exist in several fish taxa and from 5S phylogenetic relationships. An extensive analysis of NTS-II sequences of Rajidae and Dasyatidae revealed the occurrence of large simple sequence repeat (SSR) regions that are formed by microsatellite arrays. The localization and organization of SSR within the NTS-II are conserved in Rajiformes since the Upper Cretaceous. The direct correlation between the SSRs extension and the NTS length indicated that they might play a role in the maintenance of the larger 5S rDNA clusters in rays. The phylogenetic analysis indicated that NTS-II is a valuable systematic tool limited to distantly related taxa of Rajiformes.

Animals↗

PubMed1978

Conservation of the primary structure at the 3' end of 18S rRNA from eucaryotic cells.

DNA sequencing methods have been used to determine a sequence of about 20 nucleotides at the 3' termini of various 18S (small ribosomal subunit) RNA molecules. Polyadenylated rRNA was first synthesized using the enzyme ATP:polynucleotidyl transferase from mainze. Then in the presence of an oligonucleotide primer uniquely complementary to the end of each adenylated rRNA, a cDNA copy was produced using AMV reverse transcriptase. In every case, the cDNA transcript was of finite size, which we ascribe to the appearance of an oligonucleotide containing m62A near the 3' end of the 18S rRNAs. Sequences at the 3' termini of 18S rRNA molecules from the four eucaryotic species examined here (mouse, silk worm, wheat embryo and slime mold) are highly conserved. They also exhibit strong homology to the 3' end of E. coli 16S rRNA. Two important differences, however, are apparent. First, the 16S sequence CCUCC, implicated in mRNA binding by E. coli ribosomes, is absent from each eucaryotic rRNA sequence. Second, a purine-rich region which exhibits extensive complementarity to the 5' noncoding regions of many eucaryotic mRNAs appears consistently.

Animals↗

PubMed2005

Immobilized stem-loop structured probes as conformational switches for enzymatic detection of microbial 16S rRNA.

We have designed and evaluated novel DNA stem-loop structured probes for enzymatic detection of nucleic acid targets. These probes constitute a novel class of conformational switches for enzymatic activity, which in the absence of a target sterically shield an affinity label and upon hybridization of the target to the recognition sequence that forms the loop of the probe restore accessibility of the label for the binding of a reporter enzyme. Analysis of probe characteristics revealed stem stability as the most important parameter governing detection functionality, while other factors such as the length of linker molecules attaching the label to the stem-loop structure and the nature of the solid support proved to be less critical. Apparently, the bulky nature of the reporter enzyme facilitates shielding of the label in the absence of the target, thereby conferring considerable structural tolerance to the conformational switch system. The stem-loop structured probes allow sensitive detection of unlabeled nucleic acid targets. Employing a microtiter assay format, 4 ng of bacterial 16S ribosomal RNA corresponding to 8 fmol could be detected, which can be compared favorably with current immobilized molecular beacon concepts based on fluorescence detection.

Colorimetry↗

PubMed2001

Crystal structure of the transcription factor sc-mtTFB offers insights into mitochondrial transcription.

Although it is commonly accepted that binding of mitochondrial transcription factor sc-mtTFB to the mitochondrial RNA polymerase is required for specific transcription initiation in Saccharomyces cerevisiae, its precise role has remained undefined. In the present work, the crystal structure of sc-mtTFB has been determined to 2.6 A resolution. The protein consists of two domains, an N-terminal alpha/beta-domain and a smaller domain made up of four alpha-helices. Contrary to previous predictions, sc-mtTFB does not resemble Escherichia coli sigma-factors but rather is structurally homologous to rRNA methyltransferase ErmC'. This suggests that sc-mtTFB functions as an RNA-binding protein, an observation standing in contradiction to the existing model, which proposed a direct interaction of sc-mtTFB with the mitochondrial DNA promoter. Based on the structure, we propose that the promoter specificity region is located on the mitochondrial RNA polymerase and that binding of sc-mtTFB indirectly mediates interaction of the core enzyme with the promoter site.

Amino Acid Sequence↗

PubMedDate not supplied

[Primary structure of hairpin 35 and 48 small rRNA is proof that Trefusiidae is a subtaxon of marine Enoplida (Nemotoda)].

A rare nucleotide substitution was found in the evolutionarily conserved loop of hairpin 35 of the 18S rRNA gene of marine free-living nematode, Trefusia zostericola (Nematoda: Enoplida). The same substitution was found in all the marine Enoplida studied but not in other nematodes. Such a molecular synapomorphy indicates that marine enoplids are more closely related to T. zostericola than to freshwater Triplonchida. Maximum parsimony, neighbor-joining, and maximum likelihood analyses of complete nucleotide sequences of the gene, with the heterogeneity of nucleotide sites in evolution rates taken into account, support this conclusion. Hence, the hypothesis of particular primitiveness of Trefusiidae among nematodes should be rejected. Phylogenies based on molecular data support the morphological reduction of metanemes in Trefusiidae. Alongside with the unique change in hairpin 35 loop among marine Enoplida (including T. zostericola), hairpin 48 is also modified by a rare transversion which could be found among Mesorhabditoidea nematodes, in related genera Pelodera, Mesorhabditis, Teratorhabditis, Parasitorhabditis, Crustorhabditis, and Distolabrellus, and in 11 orders of Rhodophyta. Rare mutations in hairpins 35 and 48 tend to be fixed correlatively in evolution and could be found in all the Acanthocephala species. X-Ray data show that these regions (H31 and H43, in alternative nomenclature) are spatially brought together in native ribosomes. The nature and distribution of molecular autoapomorphies in phylogenetic trees of high-rank taxa are discussed.

Animals↗

PubMed2005

Primary succession of soil Crenarchaeota across a receding glacier foreland.

The development of soil archaeal community structures in relation to primary succession in bulk and rhizosphere soil was examined across the forefield of the receding Rotmoosferner glacier in Austria. Using cloning and denaturing gradient gel electrophoresis (DGGE) analysis of reverse transcription polymerase chain reaction (RT-PCR) products of extracted 16S rRNA, archaeal community structure was compared over a chronosequence representing approximately 150 years of soil development and to reference sites outside the glacier forefield, representing soil exposed for approximately 9500 years. Archaeal community composition was found to be dominated by members of the non-thermophilic or Group 1 Crenarchaeota, where a dramatic yet highly structured successional sequence was observed. Succession over the 150 years sequence could be identified as occurring in three stages, each of which had a phylogenetically distinct 1.1b crenarchaea community with those organisms present in pioneering and intermediate stages belonging to a lineage distinct from those in developed soils. Climax communities also contained organisms belonging to three other major non-thermophilic crenarchaeal lineages. Comparison of archaeal communities in the rhizosphere indicated that plant species composition was not the major driver of specific crenarchaeal populations. These results indicate the potential role of soil crenarchaea in the development of soil substrates, as well as ecological diversity within and between major Group 1 lineages.

Crenarchaeota↗

PubMed1989

Structure of synthetic unmethylated 16S ribosomal RNA as purified RNA and in reconstituted 30S ribosomal subunits.

16S ribosomal RNA was made by in vitro transcription of a cloned gene, and its structure was compared to authentic 16S ribosomal RNA. The comparison was made by subjecting the two types of 16S rRNA to chemical reagents that react specifically with unpaired bases and determining the extent of reaction by reverse transcription and gel electrophoresis of the cDNA. In solution, the rRNAs were indistinguishable in their pattern of reactivity, except for a difference at A1408 and many differences in the region between residues 470 and 562. When the 16S rRNAs were reconstituted into 30S ribosomal subunits, these reactivity differences were absent. When the synthetic 16S rRNA was reconstituted into 30S subunits and then extracted and tested in solution, its pattern of chemical reactivity in the 470-562 region was the same as authentic 16S rRNA, but differences in the 1408-1410 region persisted. This study indicates that the synthetic 16S rRNA has a secondary structure in solution similar to a native secondary structure except in two regions, one apparently incorrectly folded during synthesis and the other in which nucleotides which are normally methylated in authentic 16S rRNA may be responsible for a structural difference.

Base Sequence↗

PubMed2004

Phylogenetic relationships among marine Alteromonas-like proteobacteria: emended description of the family Alteromonadaceae and proposal of Pseudoalteromonadaceae fam. nov., Colwelliaceae fam. nov., Shewanellaceae fam. nov., Moritellaceae fam. nov., Ferrimonadaceae fam. nov., Idiomarinaceae fam. nov. and Psychromonadaceae fam. nov.

The phylogenetic relationships among marine Alteromonas-like bacteria of the genera Alteromonas, Pseudoalteromonas, Glaciecola, Thalassomonas, Colwellia, Idiomarina, Oceanimonas, Oceanisphaera, Shewanella, Moritella, Ferrimonas, Psychromonas and several other genera of the 'Gammaproteobacteria' were studied. Results of 16S rRNA gene sequence analyses revealed that some members of these genera formed several coherent groups at the family level. Characteristic signature oligonucleotides for studied taxa were defined. Signature positions are divided into three classes: (i) single compensatory mutations, (ii) double compensatory mutations and (iii) mutations affecting nucleotides not paired in the secondary structure. The 16S rRNA gene sequence similarity level within genera was 93 % or above. This value can be a useful additional criterion for genus discrimination. On the basis of this work and previous polyphasic taxonomic studies, the circumscription of the family Alteromonadaceae is limited to the genera Alteromonas and Glaciecola and the creation is proposed of the families Pseudoalteromonadaceae fam. nov. to accommodate bacteria of the genera Pseudoalteromonas and Algicola gen. nov. (formerly Pseudoalteromonas bacteriolytica) and Colwelliaceae fam. nov. to accommodate bacteria of the genera Colwellia and Thalassomonas. Bacteria of the genera Oceanimonas and Oceanisphaera formed a robust cluster and shared common signature oligonucleotides. Because of deep branching and lack of association with any other genus, the following families are proposed that include single genera: Idiomarinaceae fam. nov., Psychromonadaceae fam. nov., Moritellaceae fam. nov., Ferrimonadaceae fam. nov. and Shewanellaceae fam. nov. Finally, this study also revealed that [Hyphomicrobium] indicum should be reclassified as Photobacterium indicum comb. nov.

Alteromonas↗