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PubMed1993

5' and 3' untranslated regions of pestivirus genome: primary and secondary structure analyses.

Within the conserved 5' untranslated region (UTR) of the pestivirus genome three highly variable regions were identified. Preceding the polyprotein start codon, multiple cryptic AUG codons and several small open reading frames are characteristic for all the five pestiviruses. Inspection of the context of AUGs revealed that the polyprotein initiation AUG of pestivirus has a weak context for efficient translation initiation. The most favorable context was found in two of the cryptic AUGs. Two oligopyrimidine-rich tracts upstream to the conserved either cryptic or authentic AUG in the 5'-UTR of pestivirus were identified and 83.3% of their nucleotide sequences are complementary to the consensus sequence at the 3' terminus of eucaryotic 18S rRNA. A secondary structure model for the 5'-UTR of pestivirus was predicted. Nucleotide sequence comparison among five pestiviruses led to the identification of a variable region and a conserved region in the 3'-UTR. A deletion of 41 nucleotides was found within the variable region in Osloss. A secondary structure model for the 3'-UTR was also predicted. The structural similarity of the 5'-UTR between pestiviruses and picornaviruses and hepatitis C viruses was demonstrated and the possible implications of features of the 5' and 3'-UTR of pestiviruses are discussed.

Base Sequence↗

PubMed2000

Quantification of Hyphomicrobium populations in activated sludge from an industrial wastewater treatment system as determined by 16S rRNA analysis.

The bacterial community structure of the activated sludge from a 25 million-gal-per-day industrial wastewater treatment plant was investigated using rRNA analysis. 16S ribosomal DNA (rDNA) libraries were created from three sludge samples taken on different dates. Partial rRNA gene sequences were obtained for 46 rDNA clones, and nearly complete 16S rRNA sequences were obtained for 18 clones. Seventeen of these clones were members of the beta subdivision, and their sequences showed high homology to sequences of known bacterial species as well as published 16S rDNA sequences from other activated sludge sources. Sixteen clones belonged to the alpha subdivision, 7 of which showed similarity to Hyphomicrobium species. This cluster was chosen for further studies due to earlier work on Hyphomicrobium sp. strain M3 isolated from this treatment plant. A nearly full-length 16S rDNA sequence was obtained from Hyphomicrobium sp. strain M3. Phylogenetic analysis revealed that Hyphomicrobium sp. strain M3 was 99% similar to Hyphomicrobium denitrificans DSM 1869(T) in Hyphomicrobium cluster II. Three of the cloned sequences from the activated sludge samples also grouped with those of Hyphomicrobium cluster II, with a 96% sequence similarity to that of Hyphomicrobium sp. strain M3. The other four cloned sequences from the activated sludge sample were more closely related to those of the Hyphomicrobium cluster I organisms (95 to 97% similarity). Whole-cell fluorescence hybridization of microorganisms in the activated sludge with genus-specific Hyphomicrobium probe S-G-Hypho-1241-a-A-19 enhanced the visualization of Hyphomicrobium and revealed that Hyphomicrobium appears to be abundant both on the outside of flocs and within the floc structure. Dot blot hybridization of activated sludge samples from 1995 with probes designed for Hyphomicrobium cluster I and Hyphomicrobium cluster II indicated that Hyphomicrobium cluster II-positive 16S rRNA dominated over Hyphomicrobium cluster I-positive 16S rRNA by 3- to 12-fold. Hyphomicrobium 16S rRNA comprised approximately 5% of the 16S rRNA in the activated sludge.

Cloning, Molecular↗

PubMed2000

Novel point mutations in mitochondrial 16S rRNA gene of Chinese hamster cells.

To know the nature and mechanisms of spontaneous mutations in mitochondrial DNA (mtDNA), we determined, by direct cycle sequencing, the nucleotide sequence of the 3' terminal region of the mitochondrial 16S rRNA gene from chloramphenicol-resistant (CAP-R) mutants isolated in Chinese hamster V79 cells. Four different base substitutions were identified in common for the six CAP-R mutants. All mutations were heteroplasmic. One A to G transition was mapped at a site within the putative peptidyl transferase domain, the target region for chloramphenicol, and one G to A transition and two T to G transversions were located within the two different segments which form the stems of the hairpin loop structures attached to this key domain in the predicted secondary structure of 16S rRNA. The mutations detected in this study do not map to the same sites where CAP-R mutations were found previously in mammalian cells. Allele specific-PCR analyses revealed that all four mutations occurred on a single mutant-DNA molecule, but not on several ones independently. Together with the other previous reports, our data suggest that spontaneous mtDNA mutations may not be caused exclusively by oxidative DNA damage at least in 16S rRNA gene.

Animals↗

PubMed1989

Nucleotide sequence of 16S rRNA and phylogenetic position of the green sulfur bacterium Clathrochloris sulfurica.

The almost complete primary structure of the 16S rRNA from the green sulfur bacterium "Clathrochloris sulfurica" was determined by reverse transcriptase sequencing. Comparison of defined invariable parts of the molecule from representatives of 9 major lines of descent from the eubacterial kingdom shows C. sulfurica to be highly related to Chlorobium vibrioforme. The relationship between "Clathrochloris" and Chlorobium is in accord with the present allocation of these two genera into the family Chlorobiaceae.

Base Sequence↗

PubMed1996

5S rRNA binding ribosomal proteins from Thermus thermophilus: identification and some structural properties.

An unusual acidic ribosomal protein from Thermus thermophilus, TL5, that binds to 5S rRNA specifically and strongly, has been investigated. The N-terminal sequence of TL5 does not reveal any homology with known ribosomal proteins. Two large tryptic fragments of TL5 have been isolated and characterized. 5S rRNA protected TL5 and its unstable N-terminal fragment against trypsin action. The 5S rRNA binding ability of TL5 is probably inherent in its N-terminal part. The other 5S rRNA binding ribosomal protein from T. thermophilus, TL4, has been identified as a homolog of the ribosomal protein L5 from Escherichia coli.

Amino Acid Sequence↗

PubMed1997

In vivo analyses of upstream promoter sequence elements in the 5 S rRNA gene from Saccharomyces cerevisiae.

Upstream promoter elements of the Saccharomyces cerevisiae 5 S rRNA gene have been characterized by genomic DNase I "footprinting" and by in vivo mutational analyses using base substitutions and deletions. A high copy shuttle-vector was used to efficiently express the mutant 5 S rRNA genes in vivo and a structural mutation in the 5 S rRNA, which was previously shown to be functionally neutral but easily detected by gel electrophoresis, allowed for an accurate measure of gene expression. The results provide direct evidence for upstream regulatory elements which confirms a start site element (sse) from -1 to -8 and identifies a new independent upstream promoter element (upe) centered from about -17 to -20. In contrast to previous reports with reconstituted systems, both elements dramatically affect the efficiency of gene expression and suggest that the saturated conditions which are used in reconstituted studies mask sequence dependence; a dependency that could be physiologically significant and play a role in the regulation of 5 S rRNA expression. The footprint analyses support an extended region of protein interaction as recently observed in reconstituted systems but again provide evidence of significant structural rearrangements when the upstream sequence is changed.

Base Sequence↗

PubMed1986

A temperature-sensitive mutant in the gene rplX for ribosomal protein L24 and its suppression by spontaneous mutations in a 23S rRNA gene of Escherichia coli.

A temperature-sensitive mutant with an altered ribosomal protein L24 was analysed. Revertant analysis showed that the temperature-sensitive growth was correlated with the altered protein. A DNA segment containing the mutant rplX gene was cloned and sequenced. The GGC codon for glycine at the amino acid position 84 of the protein was found to be altered to a GAC codon for aspartic acid. By transforming the rplX mutant with a plasmid carrying the rrnB operon and by selecting for temperature-resistant transformants we obtained two spontaneous suppressor mutants in the gene for 23S rRNA. DNA sequence analysis of the region corresponding to the 5' end of the 23S rRNA showed a C to T alteration at position 33 in both mutants and an additional A to G alteration at position 466 in one of them. The results suggest intimate interaction of protein L24 and the 5' end of 23S rRNA in vivo and support a secondary structure model of the 23S rRNA which brings these mutational points into a close contact.

Base Sequence↗

PubMed1989

Compensatory mutations demonstrate that P8 and P6 are RNA secondary structure elements important for processing of a group I intron.

Compensatory mutations have been constructed which demonstrate that P8 and P6, two of nine proposed base-pairing interactions characteristic of group I introns, exist within the folded structure of the Tetrahymena thermophila rRNA intervening sequence, and that these secondary structure elements are important for splicing in E. coli and self-splicing in vitro. Two-base mutations in the 5' and 3' segments of P8 are predicted to disrupt P8 and a strong splicing-defective phenotype is observed in each case. A compensatory four-base mutation in P8 is predicted to restore pairing, and results in the restoration of splicing activity to nearly wild type levels. Thus, we conclude that P8 exists and is essential for splicing. In contrast to the strong phenotypes generally exhibited by mutations which disrupt RNA secondary structure, a two-base mutation in L8, the loop between P8[5'] and P8[3'], results in only a slight decrease in splicing activity. We also tested P6, a pairing which is proposed to consist of only two base-pairs in this intron. A two-base mutation in P6[3'] reduces splicing activity to a greater extent than does a two-base mutation in P6[5']. Comparison of the activities of these mutants and a compensatory P6 four-base mutant support the existence of P6, and suggest that the P6 pairing may be particularly important in the exon ligation step of splicing.

Animals↗

PubMed1973

A study of the thermal stability of ribosomes and biologically active subribosomal particles.

1. The ability of Escherichia coli ribosomes to function in poly(U)-directed protein synthesis was measured at elevated temperatures by using thermostable supernatant factors from Bacillus stearothermophilus. The amount of polyphenylalanine synthesized at 55 degrees C was about the same as at 37 degrees C, but the rate of synthesis was increased approximately fivefold. At 60 degrees C the activity of the ribosomes was halved. 2. E. coli ribosomes can sustain the loss of approx. 10% of the double-helical secondary structure of RNA without losing activity. 3. Within the active ribosome the double-helical secondary structure of the rRNA moiety is stabilized compared with isolated rRNA, as judged by enzymic hydrolysis and by measurements of E(260). 4. The main products, over the range 0-55 degrees C, of ribonuclease T(1) digestion of the smaller subribosomal particle of E. coli were two fragments (s(0) (20,w) 15S and 25.3S) of approximately one-quarter and three-quarters of the size of the intact molecule, revealing the presence of a ;weak spot' where intramolecular bonds appear insufficient to hold the fragments together. 5. Subribosomal particles of B. stearothermophilus were more stable to heating, by approx. 10 degrees C, than those of E. coli, and the stabilization of double-helical secondary structure of the RNA moiety was more striking. 6. Rabbit reticulocyte ribosomes were active in poly(U)-directed protein synthesis at 45 degrees C, and half the activity was lost after heating to 53 degrees C. Active subribosomal particles of rabbit reticulocytes and of oocytes of Xenopus laevis, like the bacterial subribosomal particles, underwent a conformational change to a slower-sedimenting form on heating. The temperature range of the transition depended on the species. 7. Slower-sedimenting particles, whether produced by EDTA treatment or by heating, had different ;melting' profiles compared with active subribosomal particles, providing another indication of conformational differences. 8. Comparison of the properties of the various subribosomal particles revealed greater variation in the secondary structure of the protein moieties (judged by measurement of circular dichroism) than in the secondary structure of the RNA moieties, which appeared to have features in common.

Animals↗

PubMed2005

Assessing the odd secondary structural properties of nuclear small subunit ribosomal RNA sequences (18S) of the twisted-wing parasites (Insecta: Strepsiptera).

We report the entire sequence (2864 nts) and secondary structure of the nuclear small subunit ribosomal RNA (SSU rRNA) gene (18S) from the twisted-wing parasite Caenocholax fenyesi texensis Kathirithamby & Johnston (Strepsiptera: Myrmecolacidae). The majority of the base pairings in this structural model map on to the SSU rRNA secondary and tertiary helices that were previously predicted with comparative analysis. These regions of the core rRNA were unambiguously aligned across all Arthropoda. In contrast, many of the variable regions, as previously characterized in other insect taxa, had very large insertions in C. f. texensis. The helical base pairs in these regions were predicted with a comparative analysis of a multiple sequence alignment (that contains C. f. texensis and 174 published arthropod 18S rRNA sequences, including eleven strepsipterans) and thermodynamic-based algorithms. Analysis of our structural alignment revealed four unusual insertions in the core rRNA structure that are unique to animal 18S rRNA and in general agreement with previously proposed insertion sites for strepsipterans. One curious result is the presence of a large insertion within a hairpin loop of a highly conserved pseudoknot helix in variable region 4. Despite the extraordinary variability in sequence length and composition, this insertion contains the conserved sequences 5'-AUUGGCUUAAA-3' and 5'-GAC-3' that immediately flank a putative helix at the 5'- and 3'-ends, respectively. The longer sequence has the potential to form a nine base pair helix with a sequence in the variable region 2, consistent with a recent study proposing this tertiary interaction. Our analysis of a larger set of arthropod 18S rRNA sequences has revealed possible errors in some of the previously published strepsipteran 18S rRNA sequences. Thus we find no support for the previously recovered heterogeneity in the 18S molecules of strepsipterans. Our findings lend insight to the evolution of RNA structure and function and the impact large insertions pose on genome size. We also provide a novel alignment template that will improve the phylogenetic placement of the Strepsiptera among other insect taxa.

Animals↗

PubMed2003

NMR structure of the ribosomal protein L23 from Thermus thermophilus.

The ribosomal protein L23 is a component of the large ribosomal subunit in which it is located close to the peptide exit tunnel. In this position L23 plays a central role both for protein secretion and folding. We have determined the solution structure of L23 from Thermus thermophilus. Uncomplexed L23 consists of a well-ordered part, with four anti-parallel beta-strands and three alpha-helices connected as beta-alpha-beta-alpha-beta-beta-alpha, and a large and flexible loop inserted between the third and fourth beta-strand. The observed topology is distantly related to previously known structures, primarily within the area of RNA biochemistry. A comparison with RNA-complexed crystal structures of L23 from T. thermophilus, Deinococcus radiodurans and Haloarcula marismourtui, shows that the conformation of the well-ordered part is very similar in the uncomplexed and complexed states. However, the flexible loop found in the uncomplexed solution structure forms a rigid extended structure in the complexed crystal structures as it interacts with rRNA and becomes part of the exit tunnel wall. Structural characteristics of importance for the interaction with rRNA and with the ribosomal protein L29, as well as the functional role of L23, are discussed.

Models, Molecular↗

PubMed1998

Involvement of the amino acids outside the active-site cleft in the catalysis of ricin A chain.

The A chain of ricin (RA) is a cytotoxic RNA N-glycosidase that inactivates ribosomes by depurination of the adenosine residue at position 4324 in 28S rRNA. Of the 267 amino acids in the protein, 231 could be deleted in one or another of 83 mutants, without the loss of the capacity to catalyze hydrolysis of a single specific nucleotide in rRNA [Morris, K. N. & Wool, I. G. (1992) Proc. Natl Acad. Sci. USA 89, 4869-4873]. Expression of 29 selected deletion mutants of RA in prokaryotic cell-free coupled transcription-translation reactions was carried out and the activities of the mutants were assessed by monitoring depurination of reticulocyte ribosomes. Kinetic analysis of five deletion mutants which retained detectable activity was performed. Deletion of amino acids outside the putative active-site cleft in these mutants significantly affected the catalytic rate rather than the interaction with ribosomes. From these data, the amino acids far from the active-site cleft appeared to be involved in alignment of the key residues for catalysis and interaction with the target tetraloop structure of 28S rRNA.

Amino Acid Sequence↗

PubMed1996

Diverged 5s rRNA sequences adjacent to 5s rRNA genes in the rDNA of Pythium pachycaule.

The non-transcribed spacer of the ribosomal DNA repeat of Pythium pachycaule was found to exist in two versions, one about 200 bp longer than the other. Both versions contained a conserved 5s rRNA gene in inverted orientation and a diverged copy of the 5s sequence as a tandem repeat, with a spacer of about 180 bp between them. This is the first report of multiple 5s-like sequences in the non-transcribed spacer of the rDNA of any organism. The two diverged 5s sequences differ from each other at nine positions in the 5' half of the sequence, but they have the same central 10-bp deletion and their 3' halves are identical. The diverged sequences, 5s' and 5s'', when aligned with the 5s sequence, have the same base at 63.9 and 58.8% of the positions respectively. The spacers between 5s sequences in both versions were highly conserved. The secondary structures of potential rRNA products of the diverged 5s sequences indicate that they may be pseudogenes. The relationships between the functional 5s gene and the diverged gene copies suggest that the 5s sequence family of P. pachycaule originated by duplication of an ancestral gene, followed by divergence and the introduction of heterogeneity. This work shows the potential for tandemization of 5s genes in the NTS, and may shed light on the evolutionary events that led to the transition from the rDNA-linked 5s genes found in Pythium species with filamentous zoosporangia to the unlinked tandem arrays found in Pythium species with globose zoosporangia.

Base Sequence↗

PubMed1977

[Primary structure of functions of RNA terminals].

Primary structure of tRNA, rRNA, nRNA, mRNA and viral RNA terminals has been studied. Various RNAs responsible for similar functions have been shown to possess some common features. Previously obtained data on the functional role of RNA terminal sequences, mechanisms of their biosynthesis, the stabilizing role of 5'- and 3'-sequences for the molecules of translated eukaryotic RNAs and some viral RNAs are reviewed.

Base Sequence↗

PubMed2004

The solution structure of ribosomal protein L18 from Bacillus stearothermophilus.

A medium resolution solution structure has been obtained for L18 from Bacillus stearothermophilus (BstL18), a ribosomal protein that stabilizes the tertiary structure of 5S rRNA and mediates its interaction with the rest of the large subunit. The N-terminal 22 amino acid residues of BstL18 are unstructured in solution. Its remaining 98 residues form a globular domain that has the same topology as the globular domains of other L18s, but the orientation of helices is different. This conformational peculiarity should not prevent BstL18 from functioning in the ribosome the same way as other L18s.

Geobacillus stearothermophilus↗

PubMed2002

Diversity and structure of the archaeal community in the leachate of a full-scale recirculating landfill as examined by direct 16S rRNA gene sequence retrieval.

The diversity and structure of the archaeal community in the effluent leachate from a full-scale recirculating landfill was characterized by direct 16S rRNA gene (16S rDNA) retrieval. Total-community DNA was extracted from the microbial assemblages in the landfill leachate, and archaeal 16S rDNAs were amplified with a universally conserved primer and an Archaea-specific primer. The amplification product was then used to construct a 16S rDNA clone library, and 70 randomly selected archaeal clones in the library were grouped by restriction fragment length polymorphism (RFLP) analysis. Sequencing and phylogenetic analysis of representatives from each unique RFLP type showed that the archaeal library was dominated by methanogen-like rDNAs. Represented in the kingdom of Euryarchaeota were phylotypes highly similar to the methanogenic genera Methanoculleus, Methanosarcina, Methanocorpusculum, Methanospirillum and Methanogenium, where the clone distribution was 48, 11, 3, 1 and 1, respectively. No sequences related to known Methanosaeta spp. were retrieved. Four rDNA clones were not affiliated with the known methanogenic Archaea, but instead, they were clustered with the uncultured archaeal sequences recently recovered from anaerobic habitats. Two chimeric sequences were identified among the clones analyzed.

Archaea↗

PubMed1989

Genes encoding 5S rRNA and tRNAs in the extremely thermophilic archaebacterium Methanothermus fervidus.

Methanothermus fervidus was shown to have two 5S rRNA-encoding genes linked in rRNA operons to 16S and 23S rRNA-encoding genes. Sequencing of a cloned 5S rRNA gene confirmed that M. fervidus is a member of the Methanobacteriales, although its 5S rRNA is also similar in both primary sequence and predicted secondary structure to the 5S rRNA of the non-methanogenic, but also extremely thermophilic archaebacterium, Thermococcus celer. Two clusters of tRNA genes have also been cloned and sequenced form M. fervidus. The smaller cluster, cloned in pET5401, is composed of 5'-tRNA(UGUThr)-tRNA(UGGPro)-tRNA(GUCAsp)-tRNA(UUUL ys)-3' and the larger cluster, cloned in pET5475, is composed of 5'-tRNA(GUUAsn)-tRNA(CAUMet)-tRNA(UUCGlu)-tRNA(UAGL eu)-tRNA(GUGHis)-3'. The encoded tRNAs, with the exception of the tRNA(Leu), translate abundant codons in M. fervidus. The tRNA genes do not contain introns or encode 3'-terminal CCA residues. Homologous clusters of tRNA genes have been sequenced from Methanococcus vannielii and Methanococcus voltae, so that comparisons of transcription signals, gene organizations and primary sequences can be made and features possibly related to thermostability identified. During evolution, a 5S rRNA gene appears to have been incorporated into the cluster of tRNA genes in the methanococci but not in M. fervidus.

Amino Acid Sequence↗

PubMed1992

Unfolding of the tertiary structure of specific tRNA and ribosomal 5S RNA from plants as studied with hydroxyl radicals.

Ribosomal 5S RNA is present in all eubacterial and eukaryotic ribosomes. Despite a large amount of experimental data on the primary and secondary structures of these types of molecules, details of their tertiary structure and their precise function in protein biosynthesis are still not known. Recently we have proposed a new model for the tertiary structure of plant 5S rRNA. In this study we applied the Fe(II)-mediated cleavage reaction to test the model. The data presented here provide experimental evidence that in the 5S rRNA molecule only a few nucleotides are buried in the tertiary structure. Similar experiments performed with methionine initiator tRNA gave results which imply the difference in its structure when compared with the X-ray structure of yeast tRNAPhe.

Base Composition↗