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PubMed1999

Structural equivalence in the transcribed spacers of pre-rRNA transcripts in Schizosaccharomyces pombe.

The structure of the internal transcribed spacer 2 (ITS2) in Schizosaccharomyces pombe was re-evaluated with respect to phylogenetically conserved features in yeasts, features in other transcribed spacer regions as well as the binding of transacting factors which potentially play a role in ribosomal maturation. Computer analyses and probes for nuclease protection indicate a very simple core structure consisting of a single extended hairpin which includes the interacting termini of the mature 5.8S and 25S rRNAs. Comparisons with ITS2 sequences in greatly diverging organisms indicate that the same feature also can be recognized. This is especially clear in organisms that contain very short sequences in which the putative structures are much less ambiguous. Diversity between organisms is the result of changes in hairpin length as well as the addition of branched helices. Protein binding and gel retardation studies with the S.pombe ITS2 further indicate that, as observed in the 3" external transcribed spacer (ETS) and ITS1 regions, the extended hairpin is not only the site of intermediate RNA cleavage during rRNA processing but also a site for specific interactions with one or more soluble factors. Taken together with other analyses on transcribed spacer regions, the present data suggest that the spacer regions all may act in a similar fashion, not only to organize the maturing terminal sequences, but also serve to organize specific soluble factors possibly acting with snoRNAs or in a manner which is analogous with that of the free snoRNPs.

Base Pairing↗

PubMed1999

A new version of the RDP (Ribosomal Database Project).

The Ribosomal Database Project (RDP-II), previously described by Maidak et al. [ Nucleic Acids Res. (1997), 25, 109-111], is now hosted by the Center for Microbial Ecology at Michigan State University. RDP-II is a curated database that offers ribosomal RNA (rRNA) nucleotide sequence data in aligned and unaligned forms, analysis services, and associated computer programs. During the past two years, data alignments have been updated and now include >9700 small subunit rRNA sequences. The recent development of an ObjectStore database will provide more rapid updating of data, better data accuracy and increased user access. RDP-II includes phylogenetically ordered alignments of rRNA sequences, derived phylogenetic trees, rRNA secondary structure diagrams, and various software programs for handling, analyzing and displaying alignments and trees. The data are available via anonymous ftp (ftp.cme.msu. edu) and WWW (http://www.cme.msu.edu/RDP). The WWW server provides ribosomal probe checking, approximate phylogenetic placement of user-submitted sequences, screening for possible chimeric rRNA sequences, automated alignment, and a suggested placement of an unknown sequence on an existing phylogenetic tree. Additional utilities also exist at RDP-II, including distance matrix, T-RFLP, and a Java-based viewer of the phylogenetic trees that can be used to create subtrees.

Base Sequence↗

PubMed1996

The Ribosomal Database Project (RDP).

The Ribosomal Database Project (RDP) is a curated database that offers ribosome-related data, analysis services and associated computer programs. The offerings include phylogenetically ordered alignments of ribosomal RNA (rRNA) sequences, derived phylogenetic trees, rRNA secondary structure diagrams and various software for handling, analyzing and displaying alignments and trees. The data are available via anonymous ftp (rdp.life.uiuc.edu), electronic mail (server@rdp.life.uiuc.edu), gopher (rdpgopher.life.uiuc.edu) and World Wide Web (WWW)(http://rdpwww.life.uiuc.edu/). The electronic mail and WWW servers provide ribosomal probe checking, screening for possible chimeric rRNA sequences, automated alignment and approximate phylogenetic placement of user-submitted sequences on an existing phylogenetic tree.

Computer Communication Networks↗

PubMed1997

The RDP (Ribosomal Database Project).

The Ribosomal Database Project (RDP) is a curated database that offers ribosome-related data, analysis services and associated computer programs. The offerings include phylogenetically ordered alignments of ribosomal RNA (rRNA) sequences, derived phylogenetic trees, rRNA secondary structure diagrams, and various software for handling, analyzing and displaying alignments and trees. The data are available via anonymous FTP (rdp.life.uiuc.edu), electronic mail (server@rdp.life.uiuc.edu), gopher (rdpgopher.life.uiuc.edu) and WWW (http://rdpwww.life.uiuc.edu/ ). The electronic mail and WWW servers provide ribosomal probe checking, approximate phylogenetic placement of user-submitted sequences, screening for possible chimeric rRNA sequences, automated alignment, and a suggested placement of an unknown sequence on an existing phylogenetic tree.

Animals↗

PubMed1995

Classical and novel approaches to the detection and localization of the numerous modified nucleotides in eukaryotic ribosomal RNA.

Human ribosomes contain more than 200 modified nucleotides. These are made up as follows: more than 100 2'-O-methyl groups, 10 methylated bases, about 95 pseudouridines and at least one other modification. Other mammalian sources that have been examined, as well as the lower vertebrate Xenopus laevis, show very similar patterns of nucleotide modifications, especially as revealed by oligonucleotide fingerprinting for methyl groups. Most of the methyl groups have been located along the rRNA primary structure by matching oligonucleotide sequence data to the complete sequences derived from rDNA. Nearly all of the methyls are in conserved core regions. Saccharomyces carlsbergensis ribosomes contain about 55% as many methyls as vertebrate ribosomes. The locations of most of the S carlsbergensis methyls are also known. However, of the numerous other eukaryotes whose rRNA sequences have been determined indirectly from rDNA, few have yielded detailed data on modified nucleotides. This is in part because the methods applied to vertebrate and yeast ribosomes are highly laborious and are not universally applicable. Therefore in the final part of this paper we briefly review other methods that have been applied to the detection and localization of modified nucleotides in rRNA. In particular, we outline progress towards developing a method whereby reverse transcription shows characteristic pausing at most of the 2'-O-methylation sites in human and Xenopus 18S rRNA. 2'-O-Methylation pauses are distinguishable from most other interruptions; the 2'-O-methyl pauses occur more strongly at low than at high dNTP concentration, whereas most other interruptions are independent of dNTP concentration.

Animals↗

PubMed1993

Transfer of Thiosphaera pantotropha to Paracoccus denitrificans.

Comparative sequence analysis of in vitro-amplified 16S rRNA genes of Thiosphaera pantotropha GB17T (T = type strain) and Paracoccus denitrificans LMG 4218T revealed identical 16S rRNA primary structures for the two organisms. The level of overall DNA similarity of Thiosphaera pantotropha GB17T and P. denitrificans DSM 65T is 85%, as determined by quantitative DNA-DNA hybridization. Therefore, we propose the transfer of Thiosphaera pantotropha to P. denitrificans. The closest relative of Thiosphaera pantotropha and P. denitrificans is Thiobacillus versutus, as revealed by comparative 16S rRNA sequence analysis. These organisms are members of the alpha subclass of the Proteobacteria. Within this subclass, Thiosphaera pantotropha, P. denitrificans, and Thiobacillus versutus form a phylogenetic group with Rhodobacter sphaeroides, Rhodobacter capsulatus, and "Erythrobacter longus."

Bacteria↗

PubMed1991

Conformation and structural fluctuations of a 218 nucleotides long rRNA fragment: 4-thiouridine as an intrinsic photolabelling probe.

The structure in solution of an RNA fragment (218 nucleotides long) containing part of E. coli 16S rRNA domain 2 has been studied using the intrinsic photoaffinity probe 4-thiouridine (s4U). In vitro transcription with T7 polymerase, in the presence of s4U triphosphate yielded complete RNA molecules. An affinity electrophoresis system based on Phenylmercuric substituted polyacrylamide (APM) gels allows separation of the RNA chains as a function of their s4U content. Distribution of s4U within chains follows a binomial law indicating that (i) substitution is close to random, (ii) efficiency of s4U incorporation is 0.22 times that of U. The monothiolated RNA fraction isolated from APM gel was irradiated at 366 nm under native conditions and the intramolecularly crosslinked molecules, (34%), were separated on denaturing polyacrylamide gel according to loop size. The positions of the two partners of bridges were identified by mean of reverse transcription and RNA sequencing. 17 of the 41 possible s4U positions lead to detectable bridges. These crosslinks formed efficiently at the border of bihelical regions or when structural mobility is allowed. The pattern of crosslinks is in agreement with the previously proposed secondary structure but indicates that it is much more flexible than expected.

Base Sequence↗

PubMed2004

Purine loading, stem-loops and Chargaff's second parity rule: a discussion of the application of elementary principles to early chemical observations.

DNA base compositions were determined chemically long before sequencing technologies permitted the direct counting of bases. Some recent observations made using modern sequencing technologies could have been deduced by application of elementary principles to early chemical observations. This paper draws attention to the fact that the potential for significant stem-loop structure is a general property of single-stranded DNA (genic and non-genic) and hence for any corresponding transcripts, whether they function by virtue of their structure (eg rRNA) or as mRNA. Furthermore, there is Chargaff's second parity rule: in single strands, the percentage of purines approximately equals the percentage of pyrimidines. Since, in stems, purines match pyrimidines, Szybalski's rule that transcripts violate the second parity rule in favour of purines, must apply to loops. Since purine loading occurs in both mesophilic and thermophilic species, genes with transcripts that need stable secondary structures for functioning at high temperatures must achieve this by selectively increasing the GC percentage (GC%) of stems, while retaining purine loading of loops. Arguments that purine loading is specific for the loops of RNA-synonymous strands of genes whose transcripts function by virtue of their secondary structure (ie rRNAs, not mRNAs) need to take into account, as controls, the loop regions of mRNA-synonymous strands. Entire genes, or entire genomes where gene orientation is not considered, are not appropriate controls.

Animals↗

PubMed1999

Solution structure of the ribosomal protein S19 from Thermus thermophilus.

Ribosomal protein S19 is a 10.6 kDa protein in the small subunit of the prokaryotic ribosome. We have determined a high-resolution solution structure of S19 from Thermus thermophilus. Structures were calculated using 1160 distance and dihedral angle restraints derived from (1)H, (15)N and (13)C NMR spectra. The structures show that S19 is a mixed alpha/beta protein with long disordered tails. The folding topology is not homologous to that of any other known protein structure. Potential rRNA and protein binding sites have been identified on the S19 surface.

Binding Sites↗

PubMed1989

The primary structure of the 16SrRNA of Mycobacterium leprae: its use in phylogeny and development of DNA probes.

Two long stretches of the 16S from Mycobacterium leprae were sequenced using reverses transcriptase and the chain termination technique. Homology values were calculated for 11 cultivable mycobacteria and a phylogenetic tree constructed from evolutionary distance values (Knuc). Slow and fast growing mycobacteria used in this study form a taxonomic unit but were phylogenetically well separated. It could be confirmed that M. leprae is a true member of the slowly growing pathogenic mycobacteria branching off intermediate to other members of this subgroup. Comparison of the 16 rRNA primary structures reveals that the nucleotide sequence of M. leprae contains regions of sufficient variation to serve as potential target sites for DNA probes. Here we describe the designation of a DNA oligonucleotide and its use in dot blot hybridization experiments were it was directed against bulk RNA isolated from several mycobacteria.

Base Sequence↗

PubMed1992

Phylogenetic evidence for the transfer of Eubacterium suis to the genus Actinomyces as Actinomyces suis comb. nov.

The 16S rRNA primary structures of Eubacterium suis DSM 20639T (T = type strain) and Bifidobacterium bifidum DSM 20456T were determined by sequencing in vitro amplified rDNA. Sequence comparisons indicated that B. bifidum is moderately related to representatives of the genera Actinomyces and Mobiluncus. The closest relative of E. suis is Actinomyces pyogenes. E. suis and A. pyogenes are more closely related phylogenetically to one another than to the other Actinomyces species that have been investigated by using comparative 16S rRNA analysis. Therefore, we propose that E. suis should be transferred to the genus Actinomyces as Actinomyces suis comb. nov.

Actinomyces↗

PubMed1996

Structural features of the large subunit rRNA expressed in Plasmodium falciparum sporozoites that distinguish it from the asexually expressed subunit rRNA.

The developmentally regulated transcription of at least two distinct sets of nuclear-encoded ribosomal RNAs is detected in Plasmodium species. The identification of functional differences between the two sets of rRNAs is of interest. To facilitate the search for such differences, we have identified the 5.8S and 28S rRNAs from Plasmodium falciparum that are expressed in the sporozoite stage (S gene) of the parasites' life cycle in the mosquito host and compare them to transcripts expressed in the red blood cells (A gene) of the vertebrate host. This completes the first set of A- and S-type nuclear-encoded rRNA genes for a Plasmodium species. Analysis of the predicted secondary structures of the two units reveals the majority of differences between the A- and S-type genes occur in regions previously known to be variable. However, the predicted secondary structure of both 28S rRNAs indicates 11 positions within conserved areas that are not typical of eucaryotic rRNAs. Although the A-type gene resembles almost all eucaryotes, being atypical in only 4 of the 11 positions, the S gene is variant in 8 of the 11 positions. In three of these positions, the S-type gene resembles the consensus nucleotides for the 23S rRNA from Eubacteria and/or Archaea. A few differences occur in regions associated with ribosome function, in particular the GTPase site where the S-type differs in a base pair and loop from all known sequences. Further, the identification of compensatory changes at conserved points of interactions between the 5.8S-28S rRNAs indicates that transcripts from A- and S-units should not be interchangeable.

Animals↗

PubMed1995

Pseudoknot in domain II of 23 S rRNA is essential for ribosome function.

The structure of domain II in all 23 S (and 23 S-like) rRNAs is constrained by a pseudoknot formed between nucleotides 1005 and 1138, and between 1006 and 1137 (Escherichia coli numbering). These nucleotides are exclusively conserved as 1005C.1138G and 1006C.1137G pairs in all Bacteria, Archaea and chloroplasts, whereas 1005G.1138C and 1006U.1137A pairs occur in Eukarya. We have mutagenized nucleotides 1005C-->G, 1006C-->U, 1137G-->A and 1138G-->C, both individually and in combinations, in a 23 S rRNA gene from the bacterium E. coli. The ability of 23 S rRNA to support cell growth is reduced when either of these base-pairs is disrupted, and it is completely abolished upon disruption of both base-pairs. Each mutant 23 S rRNA is assembled into 50 S subunits, but the mutant subunits do not stably interact with 30 S to engage in protein synthesis. Enzymatic and chemical probing of ribosomal particles reveals increased accessibility in the rRNA structure close to the sites of the mutations. The degree to which the mutations increase rRNA accessibility correlates with the severity of their phenotypic effects. Nucleotide 1131G is extremely reactive to dimethyl sulphate modification in wild-type subunits and ribosomes, but is rendered unreactive when either the pseudoknot is broken or when the r-proteins are removed. The structure of the pseudoknot region is possibly influenced by interaction of an r-protein at or close to the pseudoknot. Re-establishing the pseudoknot Watson-Crick interactions with one "eukaryal" (1005G.1138C or 1006U.1137A) pair and one "bacterial" C.G pair largely restores the structure and function of the rRNA. Bacterial ribosomes containing both these eukaryal pairs also participate in protein synthesis, although at much reduced efficiency, and the structure of their pseudoknot region is partially open and accessible.

Base Sequence↗

PubMed2004

Bacterial density and community structure associated with aggregate size fractions of soil-feeding termite mounds.

The building and foraging activities of termites are known to modify soil characteristics such as the heterogeneity. In tropical savannas the impact of the activity of soil-feeding termites ( Cubitermes niokoloensis) has been shown to affect the properties of the soil at the aggregate level by creating new soil microenvironments (aggregate size fractions) [13]. These changes were investigated in greater depth by looking at the microbial density (AODC) and the genetic structure (automated rRNA intergenic spacer analysis: ARISA) of the communities in the different aggregate size fractions (i.e., coarse sand, fine sand, coarse silt, fine silt, and dispersible clays) separated from compartments (internal and external wall) of three Cubitermes niokoloensis mounds. The bacterial density of the mounds was significantly higher (1.5 to 3 times) than that of the surrounding soil. Within the aggregate size fractions, the termite building activity resulted in a significant increase in bacterial density within the coarser fractions (>20 mum). Multivariate analysis of the ARISA profiles revealed that the bacterial genetic structures of unfractionated soil and soil aggregate size fractions of the three mounds was noticeably different from the savanna soil used as a reference. Moreover, the microbial community associated with the different microenvironments in the three termite mounds revealed three distinct clusters formed by the aggregate size fractions of each mound. Except for the 2-20 mum fraction, these results suggest that the mound microbial genetic structure is more dependent upon microbial pool affiliation (the termite mound) than on the soil location (aggregate size fraction). The causes of the specificity of the microbial community structure of termite mound aggregate size fractions are discussed.

Acridine Orange↗

PubMed2006

The unique 16S rRNA genes of piezophiles reflect both phylogeny and adaptation.

In the ocean's most extreme depths, pressures of 70 to 110 megapascals prevent the growth of all but the most hyperpiezophilic (pressure-loving) organisms. The physiological adaptations required for growth under these conditions are considered to be substantial. Efforts to determine specific adaptations permitting growth at extreme pressures have thus far focused on relatively few gamma-proteobacteria, in part due to the technical difficulties of obtaining piezophilic bacteria in pure culture. Here, we present the molecular phylogenies of several new piezophiles of widely differing geographic origins. Included are results from an analysis of the first deep-trench bacterial isolates recovered from the southern hemisphere (9.9-km depth) and of the first gram-positive piezophilic strains. These new data allowed both phylogenetic and structural 16S rRNA comparisons among deep-ocean trench piezophiles and closely related strains not adapted to high pressure. Our results suggest that (i) the Circumpolar Deep Water acts as repository for hyperpiezophiles and drives their dissemination to deep trenches in the Pacific Ocean and (ii) the occurrence of elongated helices in the 16S rRNA genes increases with the extent of adaptation to growth at elevated pressure. These helix changes are believed to improve ribosome function under deep-sea conditions.

Adaptation, Physiological↗

PubMed1981

Altered features in the secondary structure of Vicia faba 5.8s rRNA.

We have re-examined the nucleotide sequence of Vicia faba (broad bean) 5.8S rRNA using partial chemical degradation and a new approach to high temperature (65-80 degrees C) sequencing gels. The results indicate that the secondary structure was not completely disrupted in previous studies (Tanaka, Y., Dyer, T.A. and Brownlee, G.G. (1980) Nucleic Acid Res. 8, 1259-1272) and explain ambiguities between the nucleotide sequence and T1 ribonuclease digests. Despite this revision, estimates in the secondary structure suggest that this 5.8S rRNA differs from previously examined examples in two respects, more open conformations in both the "GC-rich" and "AU-rich" stems. The secondary structure was probed under a variety of ionic conditions using limited pancreatic and T1 ribonuclease digestion and rapid gel sequencing techniques. These studies and theoretical considerations generally supported the "burp gun" model previously proposed for all 5.8S rRNAs and were inconsistent with the recently suggested "cloverleaf" configuration. More importantly, they were also consistent with more open stem structures in this higher plant.

Animals↗

PubMed2001

Phylogenetic relationships among fruit flies, Bactrocera (Diptera, Tephritidae), based on the mitochondrial rDNA sequences.

Nucleotide sequences of a 1.6 kb long portion of the mitochondrial DNA containing the majority of the 16S rRNA gene, the tRNAval gene, and the 5' half-region of the 12S rRNA gene were determined for forty-eight individuals of nineteen Bactrocera species and one other tephritid taxon, Anastrepha ludens. Phylogenetic analyses were performed using the consistently aligned 1.5 kb long sequences, excluding seventeen portions that could not be aligned unambiguously and were aligned inconsistently among analyses using different programs and parameters. Results of phylogenetic analyses were highly congruent among distance, unweighted parsimony, and weighted parsimony methods in terms of topology of resulting trees. Bootstrap analyses supported many of the phylogenetic relationships resolved by these analyses. Although aligned sequences showed apparent biases in nucleotide content and substitutions that differed among sites within the stem/loop structure of rRNA molecules, compensation for such factors did not greatly affect the topology. The results are discussed in relation to the taxonomic positions of species used in this study and in relation to the phylogenetic and diagnostic utility of the mitochondrial rDNA fragment.

Animals↗

PubMed1986

Molecular mechanism of introduction of the hidden break into the 28S rRNA of insects: implication based on structural studies.

We determined the structure around the gap region between 28S alpha and 28S beta rRNA of Bombyx mori, a lepidopteran insect, to know the introduction mechanism of the hidden break, an interruption of the phosphodiester bond specific to the 28S rRNA of protostomes. Sequence analysis and S1 nuclease mapping suggested that a stretch of 30 nucleotides is excised from the mid region of the 28S rRNA to generate the hidden break. The length of excluded stretch was very various among three insects so far studied. However, the gap responsible for the hidden break was located in a fixed position in the 28S rRNA irrespective of insect species. It was suggested that extremely AU-rich sequence including the specific UAAU tract forming a loop can be a signal for the introduction of the hidden break. The same signal seemed also involved in splitting the dipteran 5.8S rRNA.

Animals↗