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PubMed2002

Sequence analysis and structure prediction of 23S rRNA:m1G methyltransferases reveals a conserved core augmented with a putative Zn-binding domain in the N-terminus and family-specific elaborations in the C-terminus.

N1-methylation of G748 within 23S ribosomal RNA results in resistance to the macrolide tylosin in Streptomyces. In contrast, the Escherichia coli mutant lacking N1-methylation of G745 exhibits increased resistance to viomycin, in addition to severe defects of growth characteristics. Both methylated guanines are located in hairpin 35, in domain II of prokaryotic 23S rRNA. G748 and G745 are modified by related S-adenosylmethionine-dependent methyltransferases (MTases), TlrB and RrmA respectively. Earlier sequence comparisons allowed identification of the AdoMet-binding site, however the catalytic site and the target-recognition region of these enzymes could not be delineated unambiguously. In this work, we carried out sequence-to-structure threading of the rRNA:m1G MTase family against the database of known structures to Identify those "missing regions". Our analysis confirms the earlier prediction of the AdoMet-binding site, but suggests a different location of the putative catalytic center than was previously postulated. We predict that RrmA and TlrB possess two regions that may be responsible for specific interactions with their target nucleic acid sequences: a putative Zn-finger domain in the N-terminus and the variable domain close to the C-terminus, which indicates that 23S rRNA MTases exhibit the primary structural organization distinct from other nucleic acid MTases, despite sharing the common catalytic domain.

Amino Acid Sequence↗

PubMed2003

Intra-chromosomal heterogeneity between the four 16S rRNA gene copies in the genus Veillonella: implications for phylogeny and taxonomy.

Among the seven species characterized within the genus Veillonella, three (Veillonella dispar, Veillonella parvula and Veillonella atypica) have so far been isolated from human flora and during infectious processes. Sequencing and analysis of 16S rDNA (rrs) has been described as the best method for identification of Veillonella strains at the species level since phenotypic characteristics are unable to differentiate between species. rrs sequencing for the three species isolated from humans showed more than 98 % identity between them. Four rrs copies were found in the reference strains and in all the clinical isolates studied. The sequences of each rrs were determined for the clinical strain ADV 360.1, and they showed a relatively high level of heterogeneity (1.43 %). In the majority of cases, polymorphic positions corresponded to nucleotides allowing differentiation between the three species isolated from humans. Moreover, variability observed between rrs copies was higher than that between 16S rDNA sequences of V. parvula and V. dispar. Phylogenetic analysis showed that polymorphism between rrs copies affected the position of strain ADV 360.1 in the tree. Variable positions occurred in stems and loops belonging to variable and hypervariable regions of the 16S rRNA secondary structure but did not change the overall structure of the 16S rRNA. PCR-RFLP experiments performed on 27 clinical isolates of Veillonella sp. suggested that inter-rrs heterogeneity occurs widely among the members of the genus VEILLONELLA: These results, together with the lack of phenotypic criteria for species differentiation, give preliminary arguments for unification of V. dispar and V. parvula.

Base Sequence↗

PubMed2000

Whole-cell versus total RNA extraction for analysis of microbial community structure with 16S rRNA-targeted oligonucleotide probes in salt marsh sediments.

rRNA-targeted oligonucleotide probes have become powerful tools for describing microbial communities, but their use in sediments remains difficult. Here we describe a simple technique involving homogenization, detergents, and dispersants that allows the quantitative extraction of cells from formalin-preserved salt marsh sediments. Resulting cell extracts are amenable to membrane blotting and hybridization protocols. Using this procedure, the efficiency of cell extraction was high (95.7% +/- 3.7% [mean +/- standard deviation]) relative to direct DAPI (4',6'-diamidino-2-phenylindole) epifluorescence cell counts for a variety of salt marsh sediments. To test the hypothesis that cells were extracted without phylogenetic bias, the relative abundance (depth distribution) of five major divisions of the gram-negative mesophilic sulfate-reducing delta proteobacteria were determined in sediments maintained in a tidal mesocosm system. A suite of six 16S rRNA-targeted oligonucleotide probes were utilized. The apparent structure of sulfate-reducing bacteria communities determined from whole-cell and RNA extracts were consistent with each other (r(2) = 0.60), indicating that the whole-cell extraction and RNA extraction hybridization approaches for describing sediment microbial communities are equally robust. However, the variability associated with both methods was high and appeared to be a result of the natural heterogeneity of sediment microbial communities and methodological artifacts. The relative distribution of sulfate-reducing bacteria was similar to that observed in natural marsh systems, providing preliminary evidence that the mesocosm systems accurately simulate native marsh systems.

Ecosystem↗

PubMedDate not supplied

[Primary structure of the 5S rRNA gene and its flanking sequences in diploid Aegilops tauschii Coss].

In genome of diploid aegilops A. tauschii (k773) two types of sequences 420 and 510 b. p. long which hybridize with 5S[32P]rRNA were discovered. Using the plasmid pBR327 the recombinant plasmids pAt5S79 and pAt5S91 containing 5S DNA repeats of Ae. tauschii were constructed. The primary structure of 5S rRNA gene and that of intergene spacer cloned in pAt5S79 was determined. In gene region of Ae. tauschii coding for 5S rRNA, as compared with T. aestivum, there were found 3 base substitutions in positions 24, 36 and 37. The homology of spacer sequences is 79.1%. In 5S rRNA gene of Ae. tauschii GC-content is 53.3%, but in a spacer--48.1%. The terminator of transcription in Ae. tauschii includes 15 AT-base pairs with predomination of T-bases in uncoding chain.

DNA↗

PubMed1990

New model of tertiary structure of plant 5S rRNA is confirmed by digestions with alpha-sarcin.

The cytotoxin alpha-sarcin was employed to test the model of secondary and tertiary structures of plant 5S rRNAs, which we recently proposed [(1990) Int. J. Biol. Macromol. (in press)]. alpha-Sarcin is a novel ribonuclease that hydrolyzes phosphodiester bonds adjacent to purines in nucleic acids. The digestion pattern obtained for lupin and wheat germ 5S rRNAs strongly suggests the existence of tertiary interactions between residues C34, C35, C36, A37 and G85, G86, G87, U88 as previously proposed. The results on the secondary structure of plant 5S rRNA are in line with a previously proposed model.

Base Sequence↗

PubMed1994

Nucleotide sequence and secondary structures of precursor 16S rRNA of slow-growing mycobacteria.

Slow-growing mycobacteria have a single ribosomal RNA (rrn) operon, with the genes for 16S, 23S and 5s rRNA being present in that order. The transcription start site of the rrn operon of Mycobacterium tuberculosis was identified in Escherichia coli. PCR methodology was used to amplify parts of the rrn operon, namely the leader region and the spacer-1 region separating the 16S rRNA and 23S rRNA genes of Mycobacterium avium, Mycobacterium paratuberculosis, Mycobacterium intracellulare, 'Mycobacterium lufu', Mycobacterium simiae and Mycobacterium marinum. The amplified DNA was sequenced. The sequence data, together with those obtained previously for Mycobacterium leprae and M. tuberculosis, were used to identify putative antitermination signals and RNase III processing sites within the leader region. Notable features include a highly conserved Box B element and a sequence of 31 nucleotides which is common to all eight slow-growers which were scrutinized. A secondary structure for mycobacterial precursor-16S rRNA was devised, based on sequence homologies and homologous nucleotide substitutions. The 18 nucleotides at the 5'-end of spacer-1 have the capacity of binding sequences close to the 5'- and 3'-ends of mature 16S rRNA, suggesting that secondary structure is important to the maturation process. All the slow-growers, including M. leprae, conform to the same scheme of secondary structure. The scheme proposed for M. tuberculosis is a variant of the main theme. The leader and spacer sequences may prove a useful supplement to 16S rRNA sequences in establishing phylogenetic relationships between very closely related species. 'M. lufu' appears to be a close relative of M. intracellulare.

Base Sequence↗

PubMed1999

The NMR structure of the 5S rRNA E-domain-protein L25 complex shows preformed and induced recognition.

The structure of the complex between ribosomal protein L25 and a 37 nucleotide RNA molecule, which contains the E-loop and helix IV regions of the E-domain of Escherichia coli 5S rRNA, has been determined to an overall r.m.s. displacement of 1.08 A (backbone heavy atoms) by heteronuclear NMR spectroscopy (Protein Databank code 1d6k). The interacting molecular surfaces are bipartite for both the RNA and the protein. One side of the six-stranded beta-barrel of L25 recognizes the minor groove of the E-loop with very little change in the conformations of either the protein or the RNA and with the RNA-protein interactions occurring mainly along one strand of the E-loop duplex. This minor groove recognition module includes two parallel beta-strands of L25, a hitherto unknown RNA binding topology. Binding of the RNA also induces conversion of a flexible loop to an alpha-helix in L25, the N-terminal tip of which interacts with the widened major groove at the E-loop/helix IV junction of the RNA. The structure of the complex reveals that the E-domain RNA serves as a preformed docking partner, while the L25 protein has one preformed and one induced recognition module.

Amino Acid Sequence↗

PubMed1990

Nucleotide sequence of Citrus limon 26S rRNA gene and secondary structure model of its RNA.

The complete nucleotide sequence of Citrus limon 26S rDNA has been determined. The sequence has been aligned with large ribosomal RNA (L-rRNA) sequences of Escherichia coli, Saccharomyces cerevisiae and Oryza sativa. Nine extensive expansion segments in dicot 26S rRNA relative to E. coli 23S rRNA have been identified and compared with analogous segments of monocot, yeast, amphibian and human L-rRNAs. A secondary structure model for lemon 26S rRNA has been derived based on the refined model of E. coli 23S rRNA. It has been compared with other eukaryotic L-rRNAs models in terms of location of functionally important regions. Origin and evolution of L-rRNA expansion segments are discussed.

Base Sequence↗

PubMedDate not supplied

Structure and function of 5S rRNA in the ribosome.

5S rRNA is a small RNA molecule that is a component of a ribosome from almost all living organisms. In this review, we discuss the biogenesis of 5S rRNA and its properties as an independent structural domain of a ribosome as well as the current concepts concerning the higher order structure of 5S rRNA in free state and in its complexes with ribosomal proteins and its folding in the ribosome. Special attention is paid to recent experimental approaches that have been useful in 5S rRNA studies. Our own data on topography of 5S rRNA in the ribosomes are discussed in detail. The hypothesis describing the possible functional role of 5S rRNA for ribosome functioning is discussed.

Base Sequence↗

PubMed2000

The performance of several multiple-sequence alignment programs in relation to secondary-structure features for an rRNA sequence.

The performances of five global multiple-sequence alignment programs (CLUSTAL W, Divide and Conquer, Malign, PileUp, and TreeAlign) were evaluated using part of the animal mitochondrial small subunit (12S) rRNA molecule. Conserved sequence motifs derived from an alignment based on secondary structural information were used to score how well each program aligned a data set of five vertebrate and five invertebrate taxa over a range of parameter values. All of the programs could align the motifs with reasonable accuracy for at least one set of parameter conditions, although if the whole sequence was considered, similarity to the structural alignment was only 25%-34%. Use of small gap costs generally gave more accurate results, although Malign and TreeAlign generated longer alignments when gap costs were low. The programs differed in the consistency of the alignments when gap cost was varied; CLUSTAL W, Divide and Conquer, and TreeAlign were the most accurate and robust, while PileUp performed poorly as gap cost values increased, and the accuracy of Malign fluctuated. Default settings for the programs did not give the best results, and attempting to select similar parameter values in different programs did not always result in more similar alignments. Poor alignment of even well-conserved motifs can occur if these are near sites with insertions or deletions. Since there is no a priori way to determine gap costs and because such costs can vary over the gene, alignment of rRNA sequences, particularly the less well conserved regions, should be treated carefully and aided by secondary structure and conserved motifs. Some motifs are single bases and so are often invisible to alignment programs. Our tests involved the most conserved regions of the 12S rRNA gene, and alignment of less well conserved regions will be more problematical. None of the alignments we examined produced a fully resolved phylogeny for the data set, indicating that this portion of 12S rRNA is insufficient for resolution of distant evolutionary relationships.

Algorithms↗

PubMed1988

Sequence and secondary structure of the 5' external transcribed spacer of mouse pre-rRNA.

We report the sequence of the 4006-nucleotide 5' external transcribed spacer (5'ETS) of the mouse ribosomal primary transcript. These data complete the sequence of the 13.4-kb mouse rRNA gene, thus providing a mammalian rRNA gene structure, in addition to yeast and Xenopus. The mouse 5'ETS displays a highly biased base content (very high in GC and particularly low in A), closely similar to the other transcribed spacers of the mouse ribosomal gene. This region seems to have accumulated sequence variation relatively rapidly during vertebrate evolution, with the possible insertion in rodents of sequences structurally similar to retroposons. About half the length of the mouse 5'ETS can fold into a giant and highly stable secondary structure, which is probably evolutionarily conserved in mammals and which could play an important role in the higher-order organization of mammalian pre-ribosomes.

Animals↗

PubMed1994

A new type of RNA editing. 5S ribosomal DNA transcripts are edited to mature 5S rRNA.

The primary structure of the 5S rRNA from Arabidopsis thaliana was determined. A comparison of this nucleotide sequence with that of 5S rRNA gene showed two differences. Furthermore, we compared all plant 5S rRNA and 5S rDNA sequences known to date and found that lack of colinearity is widespread among higher plant 5S rRNAs and occurred mainly in the double stranded regions. To explain this, we suggest a mechanism which converts putative products of pseudogene ("cryptogene") to mature 5S rRNA molecule. This kind of editing mechanism functions primarily in stems of 5S rRNA in order, to correct mispairing and thereby restoring the Watson-Crick base pairs. This idea could explain why so many different 5S rRNA genes or gene like sequences and only one 5S rRNA species exist in the plant cell. The editing can serve as a new mechanism of regulation of 5S rRNA synthesis in addition to transcription of 5S rRNA gene.

Base Sequence↗

PubMed2002

Secondary structure of mitochondrial 12S rRNA among fish and its phylogenetic applications.

The complete 12S ribosomal RNA(rRNA) sequences from 23 gobioid species and nine diverse assortments of other fish species were employed to establish a core secondary structure model for fish 12S rRNA. Of the 43 stems recognized, 41 were supported by at least some compensatory evidence among vertebrates. The rates of nucleotide substitution were lower in stems than in loops. This may produce less phylogenetic information in stems when recently diverged taxa are compared. An analysis of compensatory substitution shows that the percentage of covariation is 68%, and the weighting factor for phylogenetic analyses to account for the dependence of mutations should be 0.66. Different stem-loop weighting schemes applied to the analyses of phylogenetic relationships of the Gobioidei indicate that down-weighting paired regions because of nonindependence could not improve the present phylogenetic analysis. A biased nucleotide composition (adenine% [A%] > thymine% [T%], cytosine% [C%] > guanine% [G%]) in the loop regions was also observed in the mammalian counterpart. The excess of A and C in the loop regions may be because of the asymmetric mechanism of mtDNA replication, which leads to the spontaneous deamination of C and A. This process may also be responsible for a transition-transversion bias and the patterns of nucleotide substitutions in both stems and loops.

Animals↗

PubMed1985

Secondary structure of the circular form of the Tetrahymena rRNA intervening sequence: a technique for RNA structure analysis using chemical probes and reverse transcriptase.

The structure of the intervening sequence (IVS) of the Tetrahymena rRNA precursor mediates cleavage-ligation reactions that result in pre-rRNA splicing and IVS cyclization. We have developed a method for RNA structure analysis and applied it to the circular form of the IVS RNA. The native RNA was treated with dimethyl sulfate or diethyl pyrocarbonate to modify bases not involved in secondary or tertiary interactions. The RNA was then used as a template for reverse transcription. Elongation of synthetic oligodeoxynucleotide primers was found to stop (or pause) one nucleotide prior to 1-methyladenosine, 3-methylcytidine, and 7-ethoxycarbonyladenosine residues. The detection of 1-methyladenosine is particularly useful for locating single-stranded regions. After chemical cleavage of the RNA, 7-methylguanosine also could be detected. In general, the sites of modification were consistent with a previous model of the secondary structure of the linear form of the IVS RNA, a model based on enzymatic cleavage data, free energy calculations, and phylogenetic comparison. Thus, IVS RNA autocyclization does not involve major rearrangements of the secondary structure, although there is evidence for a conformational change in one region of the molecule. The methods described here should be of general use for obtaining information about structure far from the ends of RNA molecules.

Animals↗

PubMed1983

Comparative structural analysis of cytoplasmic and chloroplastic 5S rRNA from spinach.

5S rRNAs from Spinacea oleracea cytoplasmic and chloroplastic ribosomes have been subjected to digestion with the single strand specific nuclease S1 and to chemical modification of cytidines by sodium bisulphite in order to probe the RNA structure. According to these data, cytoplasmic 5S rRNA can be folded as proposed in the general eukaryotic 5S rRNA structure (1) and 5S rRNA from chloroplastides is shown to be more related to the general eubacterial structure (2).

Base Sequence↗

PubMed1989

The 5S rRNA-histone repeat in the crustacean Artemia: structure, polymorphism and variation of the 5S rRNA segment in different populations.

5S rRNA genes are linked to the histone genes in the 13 populations of the crustacean Artemia that we have studied. In all cases, two types of repeat units are found. Southern blot analysis of all populations shows that they can be grouped into three classes: a) American bisexuals; b) Eurasian bisexuals, and c) parthenogenetic organisms (all from Eurasia). Restriction analysis of a bisexual population from San Francisco Bay shows that the two repeat units are of 9.0 and 8.5 kb (with minor heterogeneities of restriction sites). In parthenogenetic organisms, the two repeat units are of approximately 12 kb. Sequencing data from the region of the 5S rRNA from the San Francisco Bay population, shows that in both types of units, the single 5S rRNA gene (315 bp in length), is located 430 bp downstream the 3' regulatory sequences of the H2A gene, the last gene in the histone cluster. We have isolated three clones that contain 5S rRNA sequences. Two of them (one from an American bisexual and the other from a parthenogenetic population) contain histone and 5S rRNA genes, both with the same transcriptional polarity. The third clone, lacking histone genes, is likely to be an orphon derived from the parthenogenetic population.

Amino Acid Sequence↗

PubMed1987

The primary structure of rat ribosomal protein L5. A comparison of the sequence of amino acids in the proteins that interact with 5 S rRNA.

The covalent structure of rat ribosomal protein L5, which associates with 5 S rRNA in the organelle, was deduced from the sequence of nucleotides in a recombinant cDNA (pL5-6-4) and confirmed from the sequences of amino acids in portions of the protein. Ribosomal protein L5, encoded by pL5-6-4, contains 296 amino acids and has a molecular weight of 34,298. However, a second recombinant cDNA, pL5-8-5, encodes a protein with an additional methionyl residue at position 236 and may be the product of a second active L5 gene. Rat L5 is homologous to yeast YL3 and to Halobacterium cutirubrum HL13, proteins that also bind to 5 S rRNA. No significant structural similarity, however, was found between rat L5 and other 5 S rRNA-binding proteins; not with a second H. cutirubrum protein HL19, nor the Escherichia coli ribosomal proteins, L5, L18, or L25, nor the Xenopus laevis transcription factor IIIA. H. cutirubrum HL19, however, has structural identity with E. coli L5 and seems to be related to yeast YL3 and, hence, may be an evolutionary link between the prokaryotic and eukaryotic 5 S rRNA-binding proteins. A group of ribosomal proteins not known to be associated with 5 S rRNA are also related to rat L5. They include: rat L39, Euglina gracilis chloroplast S7, Saccharomyces cerevisiae L31 and L46, Homo sapiens L32 and, perhaps, several others as well. There is an especially close interrelationship between rat L5, rat L39, yeast L46, human L32, and mouse L32. These results, and others, suggest that ribosomal proteins form an extended family and that L5 may contain in its structure traces of this affinity.

Amino Acid Sequence↗

PubMed1992

Trypanosoma cruzi 5S rRNA genes: molecular cloning, structure and chromosomal organization.

To further study the ribosomal RNA genetic system in Trypanosoma cruzi, the 5S rRNA gene family was characterized. We found that this gene family is reiterated about 1600 times per diploid nuclei and is mostly organized as a tandem repeat of 481 base pairs. These gene clusters were assigned to two chromosomes of about 1500 and 1400 kilobase pairs. We found that the 5S rRNA-coding region is comprised of 120 nucleotides, and contains the well-known internal control regions of eukaryotic RNA polymerase III. The two gene-spacer regions analysed exhibit a putative signal for transcription termination and six sites homologous to the consensus sequence for the binding of transcription factor Sp1.

Animals↗