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PubMed2000

[Oogenesis in turbellarians of the genus Geocentrophora studied by light and electron microscopy. V. Oocyte nuclear structures containing factors of pre-mRNA splicing and pre-rRNA processing].

An immunoelectron study of nuclear distribution of pre-mRNA splicing and pre-rRNA processing factors was carried out for oocytes of two turbellarian species: the Baikal endemic Geocentrophora wagini and a cosmopolitan G. baltica. Using monoclonal antibodies against Sm-epitope of small nuclear RNPs (snRNPs) and SR-protein SC35, it has been shown that on different stages of oocyte growth splicing factors (snRNPs and SC35) are distributed within the whole nucleus. A fibrogranular material located near heterochromatin clumps is labeled with these antibodies. A fibrillar part of this material seems to represent perichromatin fibrils. The features of intranuclear distribution of splicing factors in Geocentrophora oocyte nuclei and their ultrastructural features suggest that pre-mRNA synthesis and splicing may occur up to the end of diplotene. In Geocentrophora oocyte nuclei a few nuclear bodies (NBs) were found. Splicing factors (snRNPs and SC35) and fibrillarin were revealed in these NBs. Homology of Geocentrophora oocyte NBs to coiled bodies of oocyte and somatic cell nuclei of other animals is discussed. During diplotene, Geocentrophora oocyte nucleoli were found to lose their granular component and to change to large fibrillar structures named "postnucleoli". The postnucleoli contain both fibrillarin and non-nucleolar spliceosomal components (snRNPs and SC35). Geocentrophora oocyte postnucleoi are compared with similar structures of mammalian oocyte nuclei, taken as an example of morphological convergence of nuclear structure organization in phylogenetically distant animal species.

Animals↗
PubMed1993

Structure of recombinant ricin A chain at 2.3 A.

The plant cytotoxin ricin is a heterodimer with a cell surface binding (B) chain and an enzymatically active A chain (RTA) known to act as a specific N-glycosidase. RTA must be separated from B chain to attack rRNA. The X-ray structure of ricin has been solved recently; here we report the structure of the isolated A chain expressed from a clone in Escherichia coli. This structure of wild-type rRTA has and will continue to serve as the parent compound for difference Fouriers used to assess the structure of site-directed mutants designed to analyze the mechanism of this medically and commercially important toxin. The structure of the recombinant protein, rRTA, is virtually identical to that seen previously for A chain in the heterodimeric toxin. Some minor conformational changes due to interactions with B chain and to crystal packing differences are described. Perhaps the most significant difference is the presence in rRTA of an additional active site water. This molecule is positioned to act as the ultimate nucleophile in the depurination reaction mechanism proposed by Monzingo and Robertus (1992, J. Mol. Biol. 227, 1136-1145).

Cloning, Molecular↗
PubMed1999

Facilitation of group I splicing in vivo: misfolding of the Tetrahymena IVS and the role of ribosomal RNA exons.

Self-splicing of the group I IVS from Tetrahymena thermophila rDNA is limited by the time required for the RNA to reach its active conformation. In vitro, folding is slow because the pre-rRNA becomes kinetically trapped in inactive structures. In vivo, splicing is 50 times more rapid, implying that misfolding of the pre-rRNA is corrected. Exon mutations that inhibit self-splicing 100-fold in vitro were fully rescued when the pre-rRNA containing the IVS was expressed in E. coli. In contrast, IVS mutations that cause misfolding were only partially suppressed at 42 degrees C, and doubled the activation energy of splicing. These results suggest that intracellular folding of the pre-rRNA involves metastable intermediates similar to those observed in vitro. Precursors with natural rRNA exons were more active and less cold-sensitive than those with non-rRNA exons. This shows that the rRNA reduces misfolding of the IVS, thereby facilitating splicing of the pre-rRNA in vivo.

Animals↗
PubMed1990

Isolation and characterization of the 5S rRNA gene of Leptospira interrogans.

The gene encoding the 5S rRNA for Leptospira interrogans serovar canicola strain Moulton was isolated and sequenced. The 5S rRNA gene occurs as a single copy within the genome and encodes a 117-nucleotide-long RNA molecule. The 5S rRNA gene is flanked at both the 5' and 3' ends by regions of A + T-rich sequences, and the 5'-flanking region contains a promoter sequence. L. interrogans has a unique and remarkable organization of the 5S rRNA gene. The 5S rRNA molecule exhibits a strong similarity to typical eubacterial 5S rRNA in terms of overall secondary structure, while the primary sequence is conserved to a lesser degree. Restriction analysis of the 5S rRNA gene indicated that the DNA sequence including the 5S rRNA gene is highly conserved in the genomes of parasitic leptospires.

Base Sequence↗
PubMed1983

Improved methods for structure probing in large RNAs: a rapid 'heterologous' sequencing approach is coupled to the direct mapping of nuclease accessible sites. Application to the 5' terminal domain of eukaryotic 28S rRNA.

We have developed a combined approach for probing native structures in large RNAs. In the first method, after digestion with a structure specific nuclease, accessible sites are mapped at sequence resolution along the entire RNA molecule which is used as a template for the reverse transcriptase elongation of a 5' end labelled selected primer (coding strand of a small restriction fragment of the cloned gene). This method circumvents any prior end-labelling of RNA, a technique with major limitations for large RNAs. In the second approach, a rapid "heterologous" sequencing can be easily applied to definite domains of an RNA molecule in a variety of species (or individuals), without additional DNA cloning nor end-labelling of RNA. By taking advantage of the presence of evolutionary conserved tracts within an RNA sequence, it allows a rapid analysis of RNA folding patterns in terms of phylogenetic comparisons : when located within such a conserved tract, selected restriction fragments from a cloned gene can be used as heterologous primers for sequencing the upstream divergent region in RNAs of other species by currently available technology, i.e. reverse transcriptase elongation in the presence of chain terminator dideoxynucleotides.

Animals↗
PubMed1990

Tertiary structure and computer modeling of plant 5S ribosomal RNA.

A new model of secondary and tertiary structure of higher plant 5S rRNA is proposed. It consists of three domains. Domain alpha includes stem I and loop A; domain beta contains stems II and III and loops B and C; domain gamma consists of stems IV and V and loops D and E. We propose that the domains beta and gamma adopt RNA-A like structure due to irregularities caused by the different in size internal loops B and E and the bulges occurring in the model. A suggested bending of RNA could bring single stranded fragments of domains beta and gamma close enough to each other to allow tertiary interactions. The new model of plant 5S rRNA differs from those suggested previously for eukaryotic 5S rRNA, by arrangement of the domains beta and gamma and the base pairing scheme of domain gamma. The model is based on our results of partial digestion obtained with single and double strand specific nucleases. The experimental results were confirmed by computer aided secondary structure prediction analysis of all higher plant 5S rRNAs and computer modeling using energy minimalization approach. Further support of our model have been provided by experiments including alpha sarcin, ribonuclease H and chemical modifications.

Base Sequence↗
PubMed2004

Crystal structure of the RluD pseudouridine synthase catalytic module, an enzyme that modifies 23S rRNA and is essential for normal cell growth of Escherichia coli.

Pseudouridine (5-beta-D-ribofuranosyluracil, Psi) is the most commonly found modified base in RNA. Conversion of uridine to Psi is performed enzymatically in both prokaryotes and eukaryotes by pseudouridine synthases (EC 4.2.1.70). The Escherichia coli Psi-synthase RluD modifies uridine to Psi at positions 1911, 1915 and 1917 within 23S rRNA. RluD also possesses a second function related to proper assembly of the 50S ribosomal subunit that is independent of Psi-synthesis. Here, we report the crystal structure of the catalytic module of RluD (residues 68-326; DeltaRluD) refined at 1.8A to a final R-factor of 21.8% (R(free)=24.3%). DeltaRluD is a monomeric enzyme having an overall mixed alpha/beta fold. The DeltaRluD molecule consists of two subdomains, a catalytic subdomain and C-terminal subdomain with the RNA-binding cleft formed by loops extending from the catalytic sub-domain. The catalytic sub-domain of DeltaRluD has a similar fold as in TruA, TruB and RsuA, with the location of the RNA-binding cleft, active-site and conserved, catalytic Asp residue superposing in all four structures. Superposition of the crystal structure of TruB bound to a T-stem loop with RluD reveals that similar RNA-protein interactions for the flipped-out uridine base would exist in both structures, implying that base-flipping is necessary for catalysis. This observation also implies that the specificity determinants for site-specific RNA-binding and recognition likely reside in parts of RluD beyond the active site.

Catalytic Domain↗
PubMed1998

Nickel- and cobalt-dependent reagents identify structural features of RNA that are not detected by dimethyl sulfate or RNase T1.

Ribosomal 5S RNA presents a particular challenge to structural investigations since this polynucleotide is too large for complete NMR characterization but lacks significant tertiary structure to modulate, for example, diagnostic alkylation of guanine N7 by dimethyl sulfate. Nickel- and cobalt-dependent reagents that are sensitive to the N7 and aromatic face of guanine have now been applied to 5S rRNA (Xenopus lavis) and provide structural information that was not previously available from traditional chemical or enzymatic probes. Although G75 had repeatedly demonstrated an average reactivity with dimethyl sulfate and minimal reactivity with RNase T1, this residue was the major target of both metal-dependent reagents. Such reactivity provides crucial support for a structural model of loop E identified by prior physical, but not chemical, methods. Similarly, the tetraloop structure of loop D was more accurately reflected by the reactivity of G87 and G89 in the presence of the nickel reagent rather than in the presence of RNase T1. In addition, nickel-dependent modification of guanine residues surrounding the three-helix junction of loop A suggests an organization that is less compact than previously considered.

Animals↗
PubMed2005

Structural similarities between mRNA for the formyl peptide receptors and 18S rRNA.

Formyl peptides released by some bacteria are powerful chemoattractants and activators of mammalian granulocytes and monocytes, acting through 7-transmembrane specific formyl peptide receptors (FPRs). Three distinct segments of the formyl peptide receptor 1 (FPR1) mRNA of Man share probabilistically significant homologies with segments of the 18S rRNA which are highly conserved from Drosophila to Man. Overall, the three segments cover approximately 24% that of the 18S rRNA sequence and approximately 36% of the FPR1 sequence. The three segments are, however, arranged in different orders in the 18S rRNAs and in the FPR1 mRNA, the segment appearing in the first location in the 18S rRNAs is located at the end of the FPR1 mRNA sequence. The hypothesis is advanced that the three "conserved" segments either derive from an ancestral gene that is the forerunner of both the ribosomal 18S genes and the FPR genes or that at some stage of evolution the FPR genes derived, at least in part, from the more ancient ribosomal 18S genes. The extant 18S rRNA sequences exhibit obvious signs of a number of breaks that occurred during evolution, especially in the transition from insects to vertebrates. Some of these events may have resulted in differential rearrangements of segments in the groups of FPR genes and ribosomal 18S genes.

Animals↗
PubMed1991

Intrageneric structure of Streptococcus based on comparative analysis of small-subunit rRNA sequences.

The partial 16S rRNA sequences of 24 Streptococcus species were determined by reverse transcription. A comparative analysis of these sequences and the sequences of seven previously studied streptococcal species revealed the presence of several clusters within the genus. The clusters obtained from the sequence analysis agreed in general with the groups outlined on the basis of the results of nucleic acid hybridization studies, but there were some exceptions. The pyogenic group was extended to include Streptococcus agalactiae, S. parauberis, S. porcinus, and S. uberis. Four oral groups were discerned; these four groups centered on S. mutans, S. salivarius, S. anginosus, and S. oralis. Some species (e.g., S. suis and S. acidominimus) did not cluster with any particular group. Our findings are discussed in the context of data from other genetic and chemotaxonomic studies.

Base Sequence↗
PubMed1994

Estimation of diversity and community structure through restriction fragment length polymorphism distribution analysis of bacterial 16S rRNA genes from a microbial mat at an active, hydrothermal vent system, Loihi Seamount, Hawaii.

PCR was used to amplify (eu)bacterial small-subunit (16S) rRNA genes from total-community genomic DNA. The source of total-community genomic DNA used for this culture-independent analysis was the microbial mats from a deep-sea, hydrothermal vent system, Pele's Vents, located at Loihi Seamount, Hawaii. Oligonucleotides complementary to conserved regions in the 16S rRNA-encoding DNA (rDNA) of bacteria were used to direct the synthesis of PCR products, which were then subcloned by blunt-end ligation into phagemid vector pBluescript II. Restriction fragment length polymorphism patterns, created by using tandem tetrameric restriction endonucleases, revealed the presence of 12 groups of 16S rRNA genes representing discrete operational taxonomic units (OTUs). The rank order abundance of these putative OTUs was measured, and the two most abundant OTUs accounted for 72.9% of all of the 16S rDNA clones. Among the remaining 27.1% of the 16S rDNA clones, none of the 10 OTUs was represented by more than three individual clones. The cumulative OTU distribution for 48 bacterial 16S rDNA clones demonstrated that the majority of taxa represented in the clone library were detected, a result which we assume to be an estimate of the diversity of bacteria in the native hydrothermal vent habitat. 16S rDNA fingerprinting of individual clones belonging to particular OTUs by using an oligonucleotide probe that binds to a universally conserved region of the 16S rDNA fragments was conducted to confirm OTU specificity and 16S rDNA identity.

Bacteria↗
PubMed2002

Phylogenetic structure of the genera Flexibacter, Flexithrix, and Microscilla deduced from 16S rRNA sequence analysis.

The 16S rDNA sequences of 40 strains of 17 species in the genus Flexibacter, 5 strains of 4 species in the genus Microscilla, and 1 strain of Flexithrix dorotheae, including all type strains of approved and validated species in these genera, were determined to reveal their phylogenetic relationships. The 16S rRNA sequence analysis demonstrated the extreme heterogeneity of the genera Flexibacter and Microscilla. The strains examined diverged into 24 distinct lines of descent (1 group included both flexibacteria and flexithrix, and 1 group included both flexibacteria and microscilla) that were remote from each other at the genus level or higher. Flexibacter strains were scattered across the cytophaga-flavobacteria-bacteroides phylum and divided into 20 phylogenetic groups, and the genus Microscilla was separated into 5 groups. Flexibacter flexilis, the type species of the genus Flexibacter, and Microscilla marina, the type species of the genus Microscilla, were isolated from other organisms in their respective genera. This means that each genus should be restricted to only the type species. Flexithrix dorotheae, the type species of the genus Flexithrix, clustered with Flexibacter aggregans. The heterogeneity was found not only within genera but also within species. Flexibacter aggregans, Flexibacter aurantiacus, Flexibacter flexilis, Flexibacter roseolus, Flexibacter tractuosus, and "Microscilla sericea" each contained phylogenetically distant strains. The taxonomic concept of the genera Flexibacter, Flexithrix, and Microscilla should be reorganized in accordance with the natural relationships revealed in this study.

Bacteroides↗
PubMed1988

Conformational analysis of 16 S ribosomal RNA from Escherichia coli by scanning transmission electron microscopy.

Digitized images of molecules of 16 S rRNA from Escherichia coli, obtained by scanning transmission electron microscopy (STEM), provide quantitative structural information that is lacking in conventional electron micrographs. We have determined the morphology, total molecular mass, mass distribution within individual rRNA molecules and apparent radii of gyration. From the linear density (M/L) we have assessed the number of strands in the structural backbone of rRNA and studied the pattern of branching and folding related to the secondary and tertiary structure of rRNAs under various buffer conditions. Even in reconstitution buffer 16 S RNA did not show any resemblance to the native 30 S subunit.

Escherichia coli↗
PubMed1992

In vivo analysis of plant RNA structure: soybean 18S ribosomal and ribulose-1,5-bisphosphate carboxylase small subunit RNAs.

A method to investigate the structure of RNA molecules within intact plant tissues has been developed. The RNA structures are analyzed using dimethyl sulfate (DMS), which modifies substituents of adenine and cytosine residues within single-stranded regions of RNA molecules. Reactive sites are identified by primer extension analysis. Using this procedure, an analysis of the secondary structure of the cytoplasmic 18S ribosomal RNA in soybean seedling leaves has been completed. DMS modification data are in good agreement with the phylogenetic structure predicted for soybean 18S rRNA. However, there are a few notable exceptions where residues thought to be involved in double-stranded regions in all 18S rRNAs are strongly modified in soybean leaf samples. These data taken together with the phylogenetic structure suggest that alternate structures may exist in vivo. The further applicability of this technique is demonstrated by comparing the modification pattern obtained in vivo to that obtained in vitro for a particular mRNA molecule encoding the small subunit of ribulose-1,5-bisphosphate carboxylase. The results obtained are compared to a predicted minimum energy secondary structure. The data indicate that the conformation of RNA molecules within the cell may not be reflected in a structural analysis of purified mRNA molecules.

Base Composition↗
PubMed1997

Leader peptides of inducible chloramphenicol resistance genes from gram-positive and gram-negative bacteria bind to yeast and Archaea large subunit rRNA.

catA86 is the second gene in a constitutively transcribed, two-gene operon cloned from Bacillus pumilus . The region that intervenes between the upstream gene, termed the leader, and the catA86 coding sequence contains a pair of inverted repeat sequences which cause sequestration of the catA86 ribosome binding site in mRNA secondary structure. As a consequence, the catA86 coding sequence is untranslatable in the absence of inducer. Translation of the catA86 coding sequence is induced by chloramphenicol in Gram-positives and induction requires a function of the leader coding sequence. The leader-encoded peptide has been proposed to instruct its translating ribosome to pause at leader codon 6, enabling chloramphenicol to stall the ribosome at that site. Ribosome stalling causes destabilization of the RNA secondary structure, exposing the catA86 ribosome binding site, allowing activation of its translation. A comparable mechanism of induction by chloramphenicol has been proposed for the regulated cmlA gene from Gram-negative bacteria. The catA86 and cmlA leader-encoded peptides are in vitro inhibitors of peptidyl transferase, which is thought to be the basis for selection of the site of ribosome stalling. Both leader-encoded peptides have been shown to alter the secondary structure of Escherichia coli 23S rRNA in vitro. All peptide-induced changes in rRNA conformation are within domains IV and V, which contains the peptidyl transferase center. Here we demonstrate that the leader peptides alter the conformation of domains IV and V of large subunit rRNA from yeast and a representative of the Archaea. The rRNA target for binding the leader peptides is therefore conserved across kingdoms.

Base Sequence↗
PubMed2001

Role of the ITS2-proximal stem and evidence for indirect recognition of processing sites in pre-rRNA processing in yeast.

Eucaryotic ribosome biogenesis involves many cis-acting sequences and trans-acting factors, including snoRNAS: We have used directed mutagenesis of rDNA plasmids in yeast to identify critical sequence and structural elements within and flanking the ITS2-proximal stem. This base paired structure, present in the mature ribosome, is formed between the 5'-end of 25S and the 3'-end of 5.8S rRNAS: Previously we demonstrated that formation of this structure was critical for pre-rRNA processing in yeast. Here we show that there are no sequence-specific recognition elements within the ITS2-proximal stem, rather the structure of this stem is critical for processing. This stem cannot exceed a specific length, but there are different length restrictions for different regions within this tripartite stem. Neither the conserved unpaired nucleotides within the stem nor the sequence of the mature rRNA at the processing sites are required for processing. Collectively, these results suggest a measuring model whereby initial cleavage within ITS2 at the C2 processing site and termination of subsequent exonuclease activity yielding the mature termini are affected by the relative position of sequence and structural elements within the ITS2-proximal stem.

Base Pairing↗