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PubMed1991

Six group I introns and three internal transcribed spacers in the chloroplast large subunit ribosomal RNA gene of the green alga Chlamydomonas eugametos.

The chloroplast large subunit rRNA gene of Chlamydomonas eugametos and its 5' flanking region encoding tRNA(Ile) (GAU) and tRNA(Ala) (UGC) have been sequenced. The DNA sequence data along with the results of a detailed RNA analysis disclosed two unusual features of this green algal large subunit rRNA gene: (1) the presence of six group I introns (CeLSU.1-CeLSU.6) whose insertion positions have not been described previously, and (2) the presence of three short internal transcribed spacers that are post-transcriptionally excised to yield four rRNA species of 280, 52, 810 and 1720 nucleotides, positioned in this order (5' to 3') in the primary transcript. Together, these RNA species can assume a secondary structure that is almost identical to that proposed for the 23 S rRNA of Escherichia coli. All three internal transcribed spacers map to variable regions of primary sequence and/or potential secondary structure, whereas all six introns lie within highly conserved regions. The first three introns are inserted within the sequence encoding the 810 nucleotide rRNA species and map within domain II of the large subunit rRNA structure; the remaining introns, found in the sequence encoding the 1720 nucleotide rRNA species, lie within either domain IV or V, as is the case for all other large subunit rDNA introns that have been documented to date. CeLSU.5 and CeLSU.6 each contain a long open reading frame (ORF) of more than 200 codons. While the CeLSU.6 ORF is not related to any known ORFs, the CeLSU.5 ORF belongs to a family of ORFs that have been identified in Podospora and Neurospora mitochondrial group I introns. The finding that a polymorphic marker showing unidirectional gene conversion during crosses between C. eugametos and Chlamydomonas moewusii is located within the CeLSU.5 ORF makes it likely that this intron is a mobile element and that its ORF encodes a site-specific endonuclease promoting the transfer of the intron DNA sequence.

Amino Acid Sequence↗

PubMedDate not supplied

[Identification of RNA-recognizing modules on the surface of ribosomal proteins].

Properties of specific interaction between ribosomal proteins and ribosomal RNAs were analyzed and a method for determination of "recognizing modules" on the protein surface was proposed. The method is based on the search of protein atoms making conserved H-bonds with RNA and forming an invariant spatial structure in homologous rRNA-protein complexes and in the isolated protein. A potential of the method is demonstrated on the determination of the recognizing modules on the surfaces of ribosomal proteins S8, S15 and L5.

Bacteria↗

PubMed1998

Development and evaluation of a PCR-based assay for detection of Haemobartonella felis in cats and differentiation of H. felis from related bacteria by restriction fragment length polymorphism analysis.

The 16S rRNA gene of Haemobartonella felis was amplified by using universal eubacterial primers and was subsequently cloned and sequenced. Based on this sequence data, we designed a set of H. felis-specific primers. These primers selectively amplified a 1,316-bp DNA fragment of the 16S rRNA gene of H. felis from each of four experimentally infected cats at peak parasitemia. No PCR product was amplified from purified DNA of Eperythrozoon suis, Mycoplasma genitalium, and Bartonella bacilliformis. Blood from the experimental cats prior to infection was negative for PCR products and was greatly diminished or absent 1 month after doxycycline treatment. The overall sequence identity of this fragment varied by less than 1.0% among experimentally infected cats. By taking into consideration the secondary structure of the 16S rRNA molecule, we were able to further verify the alignment of nucleotides and quality of our sequence data. In this PCR assay, the minimum detectable number of H. felis organisms was determined to be between 50 and 704. The potential usefulness of restriction enzymes DdeI and MnlI for distinguishing H. felis from closely related bacteria was examined. This is the first report of the utility of PCR-facilitated diagnosis and discrimination of H. felis infection in cats.

Anaplasmataceae↗

PubMed1995

Cooperative assembly of proteins in the ribosomal GTPase centre demonstrated by their interactions with mutant 23S rRNAs.

The ribosomal protein L11 binds to the region of 23S rRNA associated with the GTPase-dependent steps of protein synthesis. Nucleotides 1054-1107 within this region of the Escherichia coli 23S rRNA gene were mutagenized with bisulphite. Twenty point mutations (G-->A and C-->T transitions) and numerous multiple mutations were generated. Expression of mutant 23S rRNAs in vivo shows that all the mutations detectably alter the phenotype, with effects ranging from a slight growth rate reduction to lack of viability. Temperature sensitivity is conferred by 1071G-->A and 1092C-->U substitutions. These effects are relieved by point mutations at other sites, indicating functional interconnections within the higher order structure of this 23S rRNA region. Several mutations prevent direct binding of r-protein L11 to 23S rRNA in vitro. These mutations are mainly in a short irregular stem (1087-1102) and within a hairpin loop (1068-1072), where the protein probably makes nucleotide contacts. Some of these mutations also interfere with binding of the r-protein complex L10.(L12)4 to an adjacent site on the rRNA. When added together to rRNA, proteins L10.(L12)4 and L11 bind cooperatively to overcome the effects of mutations at 1091 and 1099. The proteins also stimulate each others binding to rRNA mutated at 1087 or 1092, although in these cases binding remains clearly substoichiometric. Surprisingly, none of the mutations prevents incorporation of L11 into ribosomes in vivo, indicating that other, as yet unidentified, factors are involved in the cooperative assembly process.

Base Sequence↗

PubMed1994

The Escherichia coli ribosomal RNA leader: a structural and functional investigation.

The structure of the E. coli ribosomal RNA leader was analyzed by treatment with single and double strand specific ribonucleases and by chemical modification. The experimentally derived data together with secondary structure calculations according to minimum free energy was used to construct a secondary structure model. The binding of purified Nus proteins to the ribosomal leader RNA was further tested. Contrary to the recently reported interactions of NusB and NusE with a nut RNA sequence we obtained evidence that the presence of NusA and NusE resulted in protection against hydroxyl radical reaction of the leader nut elements boxA, boxB and boxC. The possible significance of this interaction is discussed. In the second part of the study we analyzed effects of leader mutations, which are known to affect cell growth, on the activity of ribosomes in vivo. A system was used able to distinguish the proportion of ribosomes assembled from rRNA of chromosomal origin (wild type) and plasmid origin (mutant). It turned out that the amount of 16S RNA transcribed from genes with point mutations in the leader region decreased if ribosomal pools with different translational activities were compared. High amounts of transcripts from mutant operons were present in the free ribosomal RNA and the free 30S fractions. Significantly less 16S RNA transcripts from the mutated genes were detected in the functionally active and homogeneous 70S tight couple preparations, and even less in the polysome fraction involved in active translation. The results allow a better understanding of the function of rRNA leader sequences in structure formation and correct ribosome biogenesis.

Bacterial Proteins↗

PubMed1993

High evolutionary divergence of the 5.8S ribosomal DNA in Mimulus glaucescens (Scrophulariaceae).

Ribosomal DNA sequences for the ITS 1, 5.8S, ITS 2 and adjoining regions of the 18S and 25S were obtained from Mimulus glaucescens (Scrophulariaceae) via cloned PCR products. The spacer sequences were completely unrelated to other plant taxa, although spacer lengths were approximately the same. Interestingly, the Mimulus 5.8S sequence was much more divergent than other higher-plant rDNA sequences. Consideration of the secondary structure of the 5.8S rRNA shows that most of the changes in Mimulus are compensatory and preserve the basic secondary structure of the mature RNA molecule.

Base Sequence↗

PubMed1990

16S ribosomal RNA sequence analysis for determination of phylogenetic relationship among methylotrophs.

16S ribosomal RNAs (rRNA) of 12 methylotrophic bacteria have been almost completely sequenced to establish their phylogenetic relationships. Methylotrophs that are physiologically related are phylogenetically diverse and are scattered among the purple eubacteria (class Proteobacteria). Group I methylotrophs can be classified in the beta- and the gamma-subdivisions and group II methylotrophs in the alpha-subdivision of the purple eubacteria, respectively. Pink-pigmented facultative and non-pigmented obligate group II methylotrophs form two distinctly separate branches within the alpha-subdivision. The secondary structures of the 16S rRNA sequences of 'Methylocystis parvus' strain OBBP, 'Methylosinus trichosporium' strain OB3b, 'Methylosporovibrio methanica' strain 81Z and Hyphomicrobium sp. strain DM2 are similar, and these non-pigmented obligate group II methylotrophs form one tight cluster in the alpha-subdivision. The pink-pigmented facultative methylotrophs, Methylobacterium extorquens strain AM1, Methylobacterium sp. strain DM4 and Methylobacterium organophilum strain XX form another cluster within the alpha-subdivision. Although similar in phenotypic characteristics, Methylobacterium organophilum strain XX and Methylobacterium extorquens strain AM1 are clearly distinguishable by their 16S rRNA sequences. The group I methylotrophs, Methylophilus methylotrophus strain AS1 and methylotrophic species DM11, which do not utilize methane, are similar in 16S rRNA sequence to bacteria in the beta-subdivision. The methane-utilizing, obligate group I methanotrophs, Methylococcus capsulatus strain BATH and Methylomonas methanica, are placed in the gamma-subdivision. The results demonstrate that it is possible to distinguish and classify the methylotrophic bacteria using 16S rRNA sequence analysis.

Base Sequence↗

PubMed1981

Specific binding of a prokaryotic ribosomal protein to a eukaryotic ribosomal RNA: implications for evolution and autoregulation.

Ribosomal protein L1 from the prokaryote Escherichia coli has been shown to form a specific complex with 26S ribosomal RNA from the eukaryote Dictyostelium discoideum. The segment of Dictyostelium rRNA protected from ribonuclease digestion by L1 and the corresponding region in Dictyostelium rDNA were investigated by nucleotide sequence analysis, and an analogous section in rDNA from Xenopus laevis was identified. When the L1-specific segments from eukaryotic rRNA were compared with those from prokaryotic rRNA, striking similarities in both primary and secondary structure were apparent. These conserved features suggest a common structural basis for protein recognition and indicate that such regions became fixed at a very early stage in rRNA evolution. In addition, certain structural elements of the L1 binding sites in rRNA are also found in the initial segment of the polycistronic L11-L1 mRNA, providing support for the hypothesis that L1 participates in the regulation of ribosomal protein synthesis by specific interaction with its own mRNA.

Base Sequence↗

PubMed2002

Evolution of dinoflagellate unigenic minicircles and the partially concerted divergence of their putative replicon origins.

Dinoflagellate chloroplast genes are unique in that each gene is on a separate minicircular chromosome. To understand the origin and evolution of this exceptional genomic organization we completely sequenced chloroplast psbA and 23S rRNA gene minicircles from four dinoflagellates: three closely related Heterocapsa species (H. pygmaea, H. rotundata, and H. niei) and the very distantly related Amphidinium carterae. We also completely sequenced a Protoceratium reticulatum minicircle with a 23S rRNA gene of novel structure. Comparison of these minicircles with those previously sequenced from H. triquetra and A. operculatum shows that in addition to the single gene all have noncoding regions of approximately a kilobase, which are likely to include a replication origin, promoter, and perhaps segregation sequences. The noncoding regions always have a high potential for folding into hairpins and loops. In all six dinoflagellate strains for which multiple minicircles are fully sequenced, parts of the noncoding regions, designated cores, are almost identical between the psbA and 23S rRNA minicircles, but the remainder is very different. There are two, three, or four cores per circle, sometimes highly related in sequence, but no sequence identity is detectable between cores of different species, even within one genus. This contrast between very high core conservation within a species, but none among species, indicates that cores are diverging relatively rapidly in a concerted manner. This is the first well-established case of concerted evolution of noncoding regions on numerous separate chromosomes. It differs from concerted evolution among tandemly repeated spacers between rRNA genes, and that of inverted repeats in plant chloroplast genomes, in involving only the noncoding DNA cores. We present two models for the origin of chloroplast gene minicircles in dinoflagellates from a typical ancestral multigenic chloroplast genome. Both involve substantial genomic reduction and gene transfer to the nucleus. One assumes differential gene deletion within a multicopy population of the resulting oligogenic circles. The other postulates active transposition of putative replicon origins and formation of minicircles by homologous recombination between them.

Animals↗

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↗

PubMed1998

Functional role of rRNAs in plant translation system tested with antisense strategy.

There are regions in rRNA which are evolutionary conserved and exposed on ribosomal surface. We selected in plant material (Lupinus luteus) two of them: the alpha-sarcin domain of 26S rRNA (L-rRNA) and C loop of 5S rRNA, to be further investigated using antisense oligomers as research tools. We found inhibition of the model polypeptide biosynthesis (up to 80%) due to specific hybridization of oligomers addressed to alpha-sarcin domain and loop C. Based on our results we present the evidence for the key role played by these regions of rRNAs during protein biosynthesis in plant system. According to our hypothesis, conformational changes of these two regions are synchronised and cooperative during transition of pre- to post-translocation state of the ribosome. The correlation of structure and activity of rRNA domains in translation is shown.

Base Sequence↗

PubMed2002

Functional and structural adaptations of bacterial communities growing on particulate substrates under stringent nutrient limitation.

Biomass recycle reactors (BRRs) were used as a model system to study the functional and structural adaptations of mixed bacterial communities in response to the imposition of increasingly severe nutrient limitation. BRRs were fed synthetic media containing either spinach homogenate or autoclaved yeast cells to simulate the complex mixtures of particulate carbon sources that are often present in nature. In the BRRs fed spinach homogenate, the biomass (measured as particulate protein) exhibited a physiological response similar to previous studies as detected by 40-80% reductions in respiratory potential and by relatively stable catabolic ectoenzyme activities. Concomitant adaptations in bacterial community structure were detected by PCR-DGGE and RT-PCR-DGGE of 16S rDNA and 16S rRNA fragments, respectively. The microbial community structure was dynamic even after the biomass had reached a quasi-steady state with respect to physiological measurements. In the BRRs fed yeast cells, respiratory potentials increased 2- to 5-fold during the initial portion of the BRR run and alpha-glucosidase and beta-glucosidase activities increased 2- to 4-fold. Substantial bacterial community shifts were also detected in both the rDNA and rRNA profiles, indicating that this community was also structurally dynamic. These experiments suggest that phylogenetically different bacteria sustained the functional activities in these ecosystems in response to increasingly stringent nutrient limitation.

Bacteria↗

PubMed1989

28 S ribosomal RNA in vertebrates. Locations of large-scale features revealed by electron microscopy in relation to other features of the sequences.

The 28 S rRNA from several vertebrate species, when examined by electron microscopy, is seen to contain regions of extensive secondary structure, as first reported for HeLa-cell 28 S rRNA by Wellauer & Dawid [(1973) Proc. Natl. Acad. Sci. U.S.A. 70, 2827-2831]. Here we correlate the locations of these regions, determined from the electron-microscopic data, with the primary structure of 28 S rRNA from human, mouse and Xenopus laevis determined by sequence analysis of rDNA. The secondary-structure features observed by electron microscopy correspond closely to phylogenetically variable G + C-rich regions that largely comprise the eukaryotic expansion segments in these three species. In most if not all cases the features can be identified with long G + C-rich helices deduced from sequence data. Correlations are given between the locations of the secondary-structure features and several 'landmark' restriction sites in 28 S rDNA. By correlating the locations of the rRNA methyl groups reported elsewhere [Maden (1988) J. Mol. Biol. 201, 289-314] with the present findings it is concluded that the rRNA secondary-structure features revealed by electron microscopy are largely or wholly unmethylated.

Animals↗

PubMed2000

Enhanced cleavage of RNA mediated by an interaction between substrates and the arginine-rich domain of E. coli ribonuclease E.

Endonucleolytic cutting by the essential Escherichia coli ribonuclease RNaseE has a central role in both the processing and decay of RNA. Previously, it has been shown that an oligoribonucleotide corresponding in sequence to the single-stranded region at the 5' end of RNAI, the antisense regulator of ColE1-type plasmid replication, is efficiently cut by RNaseE. Combined with the knowledge that alteration of the structure of stem-loops within complex RNaseE substrates can either increase or decrease the rate of cleavage, this result has led to the notion that stem-loops do not serve as essential recognition motifs for RNaseE, but can affect the rate of cleavage indirectly by, for example, determining the single-strandedness of the site or its accessibility. We report here, however, that not all oligoribonucleotides corresponding to RNaseE-cleaved segments of complex substrates are sufficient to direct efficient RNaseE cleavage. We provide evidence using 9 S RNA, a precursor of 5 S rRNA, that binding of structured regions by the arginine-rich RNA- binding domain (ARRBD) of RNaseE can be required for efficient cleavage. Binding by the ARRBD appears to counteract the inhibitory effects of sub-optimal cleavage site sequence and overall substrate conformation. Furthermore, combined with the results from recent analyses of E. coli mutants in which the ARRBD of RNase E is deleted, our findings suggest that substrate binding by RNaseE is essential for the normal rapid decay of E. coli mRNA. The simplest interpretation of our results is that the ARRBD recruits RNaseE to structured RNAs, thereby increasing the localised concentration of the N-terminal catalytic domain, which in turn leads to an increase in the rate of cleavage.

Amino Acid Motifs↗

PubMed1996

Secondary structures and features of the 18S, 5.8S and 26S ribosomal RNAs from the Apicomplexan parasite Toxoplasma gondii.

The two major subunits of the ribosomal RNA (rRNA) of Toxoplasma gondii, 18S and 26S, as well as 5.8S, have been sequenced and folded according to known consensus and established secondary structures. Conserved and variable nucleotide (nt) regions were identified using multiple alignments with rRNA sequences of selected organisms. The 18S rRNA showed a well conserved core structure of 48 stems and a hypervariable V4 region identified four additional stems including a pseudoknot. The 18S rRNA contained an additional helix in the V2 region located between nt 204 to 258. We noted that T. gondii 18S does not have a true V6 region, but was organized as a motif of a simple stem. T. gondii 26S had a conserved core structure of 83 stems and its expansion segments, so-called divergent domains, demonstrated a high degree of similarity with secondary structures from rRNA of dinoflagellates and ciliates. For the T. gondii 26S sequence, we found two additional stems, D3d and D3e, composed of 140 nt having a higher deltaG value. These segments are absent from the prokaryotic rRNA structures, whereas the hypervariable V4 region of the small subunit is not as variable. The well preserved structures could indicate an additional function for the eukaryotic ribosome.

Animals↗

PubMed1983

Altered structure of ribosomal RNA transcription units in hamster cells after DRB treatment.

The adenosine analog 5,6 dichloro-1-beta-ribofuranosyl benzimidazole (DRB) inhibits specifically mRNA production in eukaryotic cells, with concomitant alterations in pre-rRNA processing and nucleolar ultrastructure. We have focused our analysis on the effects of drug treatment on rRNA transcription through observations of spread ribosomal RNA (rRNA) transcription units from hamster cells. A significant increase in the size of ribosomal matrix units is paralleled by the appearance of abnormally large RNP structures containing elongating pre-rRNA. These structures correspond to nucleolar perichromatin granules which are known to appear in various conditions of cell growth when rRNA processing is altered. It is proposed that DRB-induced impairments in rRNA maturation are largely determined by alterations in the rRNA transcription process.

Animals↗

PubMed1978

Location of the 5.8S rRNA gene of Saccharomyces cerevisiae.

Direct DNA sequence analysis of Saccharomyces cerevisiae ribosomal DNA cloned in an Escherichia coli plasmid revealed part of the structural gene for 5.8S rRNA at one end of a 700-base-pair EcoRI fragment. Taken with the previously established EcoRI restriction map of the ribosomal repeat unit, this sequence establishes that the yeast 5.8S RNA segment is located between the 18S and 28S segments in the 42S rRNA precursor and in the DNA which codes for it.

Base Sequence↗

PubMed1990

Sixteen discrete RNA components in the cytoplasmic ribosome of Euglena gracilis.

We have isolated cytoplasmic ribosomes from Euglena gracilis and characterized the RNA components of these particles. We show here that instead of the four rRNAs (17-19 S, 25-28 S, 5.8 S and 5 S) found in typical eukaryotic ribosomes, Euglena cytoplasmic ribosomes contain 16 RNA components. Three of these Euglena rRNAs are the structural equivalents of the 17-19 S, 5.8 S and 5 S rRNAs of other eukaryotes. However, the equivalent of 25-28 S rRNA is found in Euglena as 13 separate RNA species. We demonstrate that together with 5 S and 5.8 S rRNA, these 13 RNAs are all components of the large ribosomal subunit, while a 19 S RNA is the sole RNA component of the small ribosomal subunit. Two of the 13 pieces of 25-28 S rRNA are not tightly bound to the large ribosomal subunit and are released at low (0 to 0.1 mM) magnesium ion concentrations. We present here the complete primary sequences of each of the 14 RNA components (including 5.8 S rRNA) of Euglena large subunit rRNA. Sequence comparisons and secondary structure modeling indicate that these 14 RNAs exist as a non-covalent network that together must perform the functions attributed to the covalently continuous, high molecular weight, large subunit rRNA from other systems.

Animals↗