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PubMed1984

A universal model for the secondary structure of 5.8S ribosomal RNA molecules, their contact sites with 28S ribosomal RNAs, and their prokaryotic equivalent.

The phylogenetic approach (ref. 1) has been utilized in construction of a universal 5.8S rRNA secondary structure model, in which about 65% of the residues exist in paired structures. Conserved nucleotides primarily occupy unpaired regions. Multiple compensating base changes are demonstrated to be present in each of the five postulated helices, thereby forming a major basis for their proof. The results of chemical and enzymatic probing of 5.8S rRNAs (ref. 13, 32) are fully consistent with, and support, our model. This model differs in several ways from recently proposed 5.8S rRNA models (ref. 3, 4), which are discussed. Each of the helices in our model has been extended to the corresponding bacterial, chloroplast and mitochondrial sequences, which are demonstrated to be positionally conserved by alignment with their eukaryotic counterparts. This extension is also made for the base paired 5.8S/28S contact points, and their prokaryotic and organelle counterparts. The demonstrated identity of secondary structure in these diverse molecules strongly suggests that they perform equivalent functions in prokaryotic and eukaryotic ribosomes.

Animals↗

PubMed1978

Sequence arrangement of the 16S and 26S rRNA genes in the pathogenic haemoflagellate Leishmania donovani.

Kinetic and chemical analysis show that the haploid genome of Leishmania donovani has between 4.6 and 6.5 X 10(7) Kb pairs of DNA. Cot analysis shows that the genome contains 12% rapidly reassociating DNA, U3% middle repetitive DNA with an average reiteration frequency of 77 and 62% single copy DNA. Saturation hybridization experiments show that 0.82% of the nuclear DNA is occupied by rRNA coding sequences. The average repetition frequency of these sequences is determined to be 166. Sedimentation velocity studies indicate the two major rRNA species have sedimentation values of 26S and 16S, respectively. The arrangement of the rRNA genes and their spacer sequences on long strands of purified rDNA has been determined by the examination of the structure of rRNA:DNA hybrids prepared for electron microscopy by the gene 32-ethidium bromide technique. Long DNA strands are observed to contain several gene sets (16S + 26S). One repeat unit contains the following sequences in the order given: (a) A 16S gene of length 2.12 Kb, (b) An internal transcribed spacer (Spl) of length 1.23 Kb, which contains a short sequence that may code for a 5.8S rRNA, (C) 26S gene with a length of 4.31 Kb which contains an internal gap region of length 0.581 Ib, (d) An external spacer of average length 5.85 Kb.

Animals↗

PubMed1989

Cloning and sequence analysis of the 18 S ribosomal RNA gene of tomato and a secondary structure model for the 18 S rRNA of angiosperms.

The gene of a cytoplasmic 18 S ribosomal RNA (18 S rDNA) of the dicotyledonous plant tomato (Lycopersicon esculentum) cv. Rentita has been cloned, and its complete primary structure has been determined. The tomato 18 S rDNA is 1805 bp long with a G + C content of 49.6%. Its sequence exhibits 94%-96% positional identity when it is colinearly aligned with the previously reported sequences of the 17-18 S rDNAs of the dicot soybean and the monocots maize and rice. A model of the secondary structure of the 18 S rRNA of angiosperms is presented and its genera-specific structural features are compared with a current eukaryotic 18 S rRNA consensus model.

Base Sequence↗

PubMed2006

Use of 16S rRNA and rpoB genes as molecular markers for microbial ecology studies.

Several characteristics of the 16S rRNA gene, such as its essential function, ubiquity, and evolutionary properties, have allowed it to become the most commonly used molecular marker in microbial ecology. However, one fact that has been overlooked is that multiple copies of this gene are often present in a given bacterium. These intragenomic copies can differ in sequence, leading to identification of multiple ribotypes for a single organism. To evaluate the impact of such intragenomic heterogeneity on the performance of the 16S rRNA gene as a molecular marker, we compared its phylogenetic and evolutionary characteristics to those of the single-copy gene rpoB. Full-length gene sequences and gene fragments commonly used for denaturing gradient gel electrophoresis were compared at various taxonomic levels. Heterogeneity found between intragenomic 16S rRNA gene copies was concentrated in specific regions of rRNA secondary structure. Such "heterogeneity hot spots" occurred within all gene fragments commonly used in molecular microbial ecology. This intragenomic heterogeneity influenced 16S rRNA gene tree topology, phylogenetic resolution, and operational taxonomic unit estimates at the species level or below. rpoB provided comparable phylogenetic resolution to that of the 16S rRNA gene at all taxonomic levels, except between closely related organisms (species and subspecies levels), for which it provided better resolution. This is particularly relevant in the context of a growing number of studies focusing on subspecies diversity, in which single-copy protein-encoding genes such as rpoB could complement the information provided by the 16S rRNA gene.

Bacteria↗

PubMed1988

Primary and secondary structure of the 18S ribosomal RNA of the bird spider Eurypelma californica and evolutionary relationships among eukaryotic phyla.

The primary structure of the 18S rRNA of the bird spider Eurypelma californica has been determined in the framework of a study of metazoan phylogeny on the basis of ribosomal RNA structure. A secondary-structure model was derived by comparison of the sequence with that of 43 other eukaryotic small-ribosomal-subunit RNA sequences presently available. This comparison allows a rather detailed secondary-structure pattern to be postulated for a eukaryote-specific area of highly variable sequence and length for which no consensus model has hitherto been attained. A dendrogram, reflecting evolutionary relationships among the 40 eukaryotic species of known 18S rRNA structure, was constructed by a matrix method selecting the best-fitting tree on the basis of a least-squares criterion. The tree shows an early divergence of a microsporidium, an euglenoid, kinetoplastids and a slime mold. Among the remaining species, two main clusters are distinguishable, one comprising the Ciliata, the other comprising Metazoa, green plants, fungi and several protists. Among the Metazoa, the three phyla presently investigated, viz. Chordata, Arthropoda and Nemathelminthes, are distinguishable as three separate lines of descent.

Animals↗

PubMed1981

Structure of the Tetrahymena pyriformis rRNA gene. Nucleotide sequence of the transcription termination region.

The 3'-terminal regions of the 25 S rRNA and 35 S rRNA were mapped in the HindIIIC fragment of the T. pyriformis rRNA gene (Niles, E. G., and Jain, R. K. (1981) Biochemistry 20, 905-909). The precise locations of these termini were identified by S1 nuclease mapping. The 35 S rRNA was shown to contain two major 3'-ends, one identical to the 3'-end of the mature 25 S rRNA and one which is 15 bases longer. The nucleotide sequence of 440 base pairs of DNA in the terminator region was determined. There are several oligo T stretches and regions of dyad symmetry. Three models of transcription termination are presented which are based on possible secondary structures in the 3'-terminal region of the 35 S rRNA.

Animals↗

PubMed1988

The RNA N-glycosidase activity of ricin A-chain. The characteristics of the enzymatic activity of ricin A-chain with ribosomes and with rRNA.

Ricin A-chain cleaves the N-glycosidic bond at A-4324 in 28 S rRNA when intact rat ribosomes are the substrate. Cleavage occurs at a concentration of the toxin of 1 X 10(-10) M, and specificity for this single residue is retained when the concentration is as high as 3 X 10(-7) M. The apparent Michaelis constant (Km) for the reaction is 2.6 microM, and the turnover number (Kcat) is 1777 min-1. The same N-glycosidic bond is cleaved by ricin A-chain in naked 28 S rRNA, but at a greatly reduced rate. The Km value for this reaction is 5.8 microM. The results suggest that the A-chain has a similar affinity for 28 S rRNA in ribosomes and in the absence of ribosomal proteins. Ricin A-chain has no effect on 23 S rRNA in Escherichia coli ribosomes, however, the N-glycosidic bond at A-2600 in naked 23 S rRNA is cleaved by the toxin; this corresponds to the ricin site in eukaryotic 28 S rRNA. Since the Km value (3.3 microM) for the reaction with E. coli 23 S rRNA approximates that obtained with rat liver ribosomes, it is possible that E. coli ribosomal protein(s) protect this site against ricin attack in intact ribosomes. Ricin A-chain also acted on naked 16 S rRNA cleaving the N-glycosidic bond of adenine at position 1014. The results suggest that ricin A-chain recognizes a specific structure in rRNA, perhaps a loop and stem having the sequence GAGA in the loop.

Animals↗

PubMed2003

The 5' end structure of transcripts derived from the rRNA gene and the RNA polymerase I transcribed protein coding genes in Trypanosoma brucei.

Due to trans-splicing and polycistronic transcription, the 5' end structure of precursor RNAs of protein coding genes in Trypanosoma brucei has not yet been characterized. In eukaryotes, in general, the 5' ends of transcripts generated by RNA polymerase (pol) I and pol II are different. Pol I derived precursor RNAs contain an unmodified tri- or diphosphate group at their 5' ends. In contrast, pol II primary transcripts, the 5' triphosphate (initially also part of the pre-mRNA) is rapidly modified by the addition of methylated guanosine triphosphate, immediately after transcription initiation. We determined the 5' end structure of precursor RNAs of the rRNA gene and the RNA pol I transcribed protein coding gene by the differential display of RNA ligase mediated amplification of cDNA ends (DDRLACE) method. Comparing the ability of the 5' end of RNA transcripts to ligate with an RNA primer following different pre-treatments, the structure of the 5' end of RNA transcripts was characterized. We found that: (1). the 5' end of putative precursor RNAs from a pol I transcribed protein coding gene and the rRNA gene was uncapped; (2). approximately 20% of the putative rRNA precursor contained a 5' tri- or diphosphate group, representing the primary transcript and approximately 80% of the putative rRNA precursor were dephosphorylated and contained a 5' hydroxyl group; (3). the majority of putative neomycin resistance gene precursor RNAs, driven by the procyclin gene promoter (a pol I promoter), contained a 5' hydroxyl group. The procyclin-neo primary transcript, as being those containing a 5' tri- or diphosphate, was below a detectable level in the steady state RNA; and (4). we did not detect pol I transcribed precursor RNAs that contained a 5' monophosphate group. The observation that the putative pre-RNAs derived from the procyclin gene promoter, similar to those of rRNA do not have a 5' capped structure, is consistent with the notion that transcription of pol I transcribed protein coding genes is crucially dependent on trans-splicing for the cap addition.

5' Untranslated Regions↗

PubMed2000

Bacillus siralis sp. nov., a novel species from silage with a higher order structural attribute in the 16S rRNA genes.

A novel bacterial strain (171544T) was recently isolated from silage and was classified in the genus Bacillus by 16S rDNA sequence analysis. Additional silage samples have been investigated in the present study and four organisms resembling strain 171544T were isolated. Phenotypic and genotypic characterization of these bacteria showed that they constitute a new species of the genus Bacillus. This taxon was positioned in the family Bacillaceae on the basis of evolutionary distance trees using 16S rDNA sequences. Bacillus circulans, Bacillus firmus and Bacillus benzoevorans were the most closely related species with 165 rDNA similarities of 97.2, 96.3 and 95.9%, respectively. All five silage isolates shared a higher order structural feature in the 3' region of the 16S rRNA gene comprising an extension to helix 49 of 24 bp and highly similar random amplified polymorphic DNA patterns that distinguished them from the type strains of B. circulans and B. firmus. Moreover, they possessed a unique pattern of phenotypic features including subterminally or terminally located endospores which distinctly swelled the sporangium, strictly aerobic metabolism but with the ability to utilize nitrate as a terminal electron acceptor under anaerobic conditions, and hydrolysis of casein but not starch. The name Bacillus siralis is therefore proposed for this new taxon. The type strain of B. siralis is strain 171544T (= NCIMB 13601T = CIP 106295T).

Bacillus↗

PubMed2002

Rlp7p is associated with 60S preribosomes, restricted to the granular component of the nucleolus, and required for pre-rRNA processing.

Many analyses have examined subnucleolar structures in eukaryotic cells, but the relationship between morphological structures, pre-rRNA processing, and ribosomal particle assembly has remained unclear. Using a visual assay for export of the 60S ribosomal subunit, we isolated a ts-lethal mutation, rix9-1, which causes nucleolar accumulation of an Rpl25p-eGFP reporter construct. The mutation results in a single amino acid substitution (F176S) in Rlp7p, an essential nucleolar protein related to ribosomal protein Rpl7p. The rix9-1 (rlp7-1) mutation blocks the late pre-RNA cleavage at site C2 in ITS2, which separates the precursors to the 5.8S and 25S rRNAs. Consistent with this, synthesis of the mature 5.8S and 25S rRNAs was blocked in the rlp7-1 strain at nonpermissive temperature, whereas 18S rRNA synthesis continued. Moreover, pre-rRNA containing ITS2 accumulates in the nucleolus of rix9-1 cells as revealed by in situ hybridization. Finally, tagged Rlp7p was shown to associate with a pre-60S particle, and fluorescence microscopy and immuno-EM localized Rlp7p to a subregion of the nucleolus, which could be the granular component (GC). All together, these data suggest that pre-rRNA cleavage at site C2 specifically requires Rlp7p and occurs within pre-60S particles located in the GC region of the nucleolus.

Amino Acid Sequence↗

PubMed1986

The evolution of eukaryotic ribosomal DNA.

Mutations occur randomly throughout the ribosomal DNA (rDNA) sequence. Molecular drive (unequal crossing-over, gene conversion, and transposition) spreads these variations through the multiple copies of rDNA. Forces of selection act upon the variants to favor and fix them or disfavor and eliminate them. Selection has not permitted changes in regions within rRNA vital for its function; these sequences are evolutionarily conserved between diverse species. Possible functions for some of these conserved sequences are discussed. The secondary structure of rRNA is also highly conserved during evolution. However, eukaryotic rRNA is larger than prokaryotic rRNA due to blocks of "expansion segments". Arguments are put forward that expansion segments might not play any functional role. Other examples are reviewed of rDNA sequence insertion or deletion, including introns and the internal transcribed spacer 2.

Animals↗

PubMed2002

An empirical analysis of mt 16S rRNA covarion-like evolution in insects: site-specific rate variation is clustered and frequently detected.

The structural and functional analysis of rRNA molecules has attracted considerable scientific interest. Empirical studies have demonstrated that sequence variation is not directly translated into modifications of rRNA secondary structure. Obviously, the maintenance of secondary structure and sequence variation are in part governed by different selection regimes. The nature of those selection regimes still remains quite elusive. The analysis of individual bacterial models cannot adequately explore this topic. Therefore, we used primary sequence data and secondary structures of a mitochondrial 16S rRNA fragment of 558 insect species from 15 monophyletic groups to study patterns of sequence variation, and variation of secondary structure. Using simulation studies to establish significance levels of change, we found that despite conservation of secondary structure, the location of sequence variation within the conserved rRNA structure changes significantly between groups of insects. Despite our conservative estimation procedure we found significant site-specific rate changes at 56 sites out of 184. Additionally, site-specific rate variation is somewhat clustered in certain helices. Both results confirm what has been predicted from an application of non-stationary maximum likelihood models to rRNA sequences. Clearly, constraints on sequence variation evolve and leave footprints in the form of evolutionary plasticity in rRNA sequences. Here, we show that a better understanding of the evolution of rRNA sequences can be obtained by integrating both phylogenetic and structural information.

Animals↗

PubMed1999

Human Nopp140, which interacts with RNA polymerase I: implications for rRNA gene transcription and nucleolar structural organization.

Nopp140 is thought to shuttle between nucleolus and cytoplasm. However, the predominant nucleolar localization of Nopp140 homologues from different species suggests that Nopp140 is also involved in events occurring within the nucleolus. In this study, we demonstrated that the largest subunit of RNA polymerase I, RPA194, was coimmunoprecipitated with the human Nopp140 (hNopp140). Such an interaction is mediated through amino acids 204 to 382 of hNopp140. By double immunofluorescence, hNopp140 was colocalized with RNA polymerase I at the rDNA (rRNA genes) transcription active foci in the nucleolus. These results suggest that Nopp140 can interact with RNA polymerase I in vivo. Transfected cells expressing the amino-terminal half of hNopp140, hNopp140N382 (amino acids 1 to 382), displayed altered nucleoli with crescent-shaped structures. This phenotype is reminiscent of the segregated nucleoli induced by actinomycin D treatment, which is known to inhibit rRNA synthesis. Consistently, the hNopp140N382 protein mislocalized the endogenous RNA polymerase I and shut off cellular rRNA gene transcription as revealed by an in situ run-on assay. These dominant negative effects of the mutant hNopp140N382 suggest that Nopp140 plays an essential role in rDNA transcription. Interestingly, ectopic expression of hNopp140 to a very high level caused the formation of a transcriptionally inactive spherical structure occupying the entire nucleolar area which trapped the RNA polymerase I, fibrillarin, and hNopp140 but excluded the nucleolin. The mislocalizations of these nucleolar proteins after hNopp140 overexpression imply that Nopp140 may also play roles in maintenance of nucleolar integrity.

Animals↗

PubMed1998

Small-subunit ribosomal DNA of an ectomycorrhizal fungus tricholoma matsutake: sequence, structure and phylogenetic analysis.

A 2861 bp nucleotide sequence containing Tricholoma matsutake SSU rRNA gene and its flanking regions was determined and analyzed. Comparison with known SSU rDNA sequences and primer extension analysis revealed that the SSU rRNA coding region and intergenic spacer 2 (IGS2) are 1805 bp and 1043 bp in length, respectively. The IGS2 has an imperfect direct repeat (type 1) homologous to the region downstream to the 5S rRNA gene and the three imperfect tandem direct repeats (type 2) upstream to the SSU rRNA-encoding sequence. Structural analysis by a comparative method showed that the overall secondary structure of the SSU rRNA is generally similar to that of S. cerevisiae, whereas the secondary structures of the V4 regions predicted by the thermodynamics-based method showed different configurations between distantly related taxa, implying that these structural differences can provide phylogenetically informative features. Phylogenetic trees based on both the aligned SSU rRNA sequences with almost full-length sequences and V4 sequences revealed that T. matsutake is very closely related to other basidiomycetes belonging to Agaricales. Thus we propose that the V4 region is also a good source for the inference of phylogeny to support the SSU rRNA phylogeny.

Base Sequence↗

PubMed1995

Birth of the snoRNPs: the evolution of RNase MRP and the eukaryotic pre-rRNA-processing system.

The ribonucleoprotein particle RNase MRP is required for the processing of yeast pre-ribosomal RNA (pre-rRNA). A structurally related particle, RNase P, is universally required for processing of pre-tRNA, but in bacteria and archaea also cleaves a site in the pre-rRNA. This suggests that RNase MRP may have arisen in eukaryotes as a form of RNase P specialized for pre-rRNA processing. Other eukaryotic small nucleolar RNAs may have arisen as trans-acting factors that functionally replace cis-acting pre-rRNA interactions in bacteria and archaea.

Biological Evolution↗

PubMed2001

Binding interactions between the active center cleft of recombinant pokeweed antiviral protein and the alpha-sarcin/ricin stem loop of ribosomal RNA.

Pokeweed antiviral protein (PAP) is a ribosome-inactivating protein that catalytically cleaves a specific adenine base from the highly conserved alpha-sarcin/ricin loop of the large ribosomal RNA, thereby inhibiting protein synthesis at the elongation step. Recently, we discovered that alanine substitutions of the active center cleft residues significantly impair the depurinating and ribosome inhibitory activity of PAP. Here we employed site-directed mutagenesis combined with standard filter binding assays, equilibrium binding assays with Scatchard analyses, and surface plasmon resonance technology to elucidate the putative role of the PAP active center cleft in the binding of PAP to the alpha-sarcin/ricin stem loop of rRNA. Our findings presented herein provide experimental evidence that besides the catalytic site, the active center cleft also participates in the binding of PAP to the target tetraloop structure of rRNA. These results extend our recent modeling studies, which predicted that the residues of the active center cleft could, via electrostatic interactions, contribute to both the correct orientation and stable binding of the substrate RNA molecules in PAP active site pocket. The insights gained from this study also explain why and how the conserved charged and polar side chains located at the active center cleft of PAP and certain catalytic site residues, that do not directly participate in the catalytic deadenylation of ribosomal RNA, play a critical role in the catalytic removal of the adenine base from target rRNA substrates by affecting the binding interactions between PAP and rRNA.

Animals↗

PubMed1998

Performance of 18S rRNA in littorinid phylogeny (Gastropoda: Caenogastropoda).

In the past, 18S rRNA sequences have proved to be very useful for tracing ancient divergences but were rarely used for resolving more recent ones. Moreover, it was suggested that the molecule does not contain useful information to resolve divergences which took place during less than 40 Myr. The present paper takes littorinid phylogeny as a case study to reevaluate the utility of the molecule for resolving recent divergences. Two data sets for nine species of the snail family Littorinidae were analyzed, both separately and combined. One data set comprised 7 new complete 18S rRNA sequences aligned with 2 published littorinid sequences; the other comprised 12 morphological, 1 biochemical, and 2 18S rRNA secondary structure characters. On the basis of its ability to confirm generally accepted relationships and the congruence of results derived from the different data sets, it is concluded that 18S rRNA sequences do contain information to resolve "rapid" cladogenetic events, provided that they occurred in the not too distant past. 18S rRNA sequences yielded support for (1) the branching order (L. littorea, (L. obtusata, (L. saxatilis, L. compressa))) and (2) the basal position of L. striata in the Littorina clade.

Animals↗

PubMed1977

Sequence arrangement of the rDNA of Drosophila melanogaster.

The sequence arrangement of genes coding for stable rRNA species and of the interspersed spacers on long single strands of rDNA purified from total chromosomal DNA of Drosophila melanogaster has been determined by a study of the structure of rRNA:DNA hybrids which were mounted for electron microscope observation by the gene 32-ethidium bromide technique. One repeat unit contains the following sequences in the order given. First, an 18 S gene of length 2.13 +/- 0.17 kb. Second, an internal transcribed spacer (Spl) of length 1.58 +/- 0.15 kb. A short sequence coding for the 5.8S and perhaps the 2S rRNA species is located within this spacer. Third, the 28S gene with a length of 4.36 +/- 0.23 kb. About 55% of the 28S genes are unbroken or continuous (C genes). However, about 45% of the 28S genes contain an insertion of an additional segment of DNA that is not complementary to rRNA (l genes). The insertion occurs at a reproducible point 2.99 +/- 0.26 kb from the junction with Spl. The insertions are heterogeneous in length and occur in three broad size classes: 1.42 +/- 0.47, 3.97 +/- 0.55, and 6.59 +/- 0.62 kb. Fourth, an external spacer between the 28S gene and the next 18S gene which is presumably mainly nontranscribed and which has a heterogeneous length distribution with a mean length and standard deviation of 5.67 +/- 1.92 kb. Short inverted repeat stems (100-400 nucleotide pairs) occur at the base of the insertion. It is known from other studies that I genes occur only on the X chromosome. The present study shows that the I and C genes on the X chromosomes are approximately randomly assorted. The sequence arrangement on the plasmid pDm103 containing one repeat of rDNA (Glover et al., 1975) has been determined by similar methods. The I gene on this plasmid contains an inverted repeat stem. The occurrence of inverted repeat sequences flanking the insertion supports the speculation that these sequences are translocatable elements similar to procaryotic translocons.

DNA↗