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PubMed1998

Common secondary structures for the second internal transcribed spacer pre-rRNA of two subfamilies of trichostrongylid nematodes.

Sequences of the second internal transcribed spacer ribosomal DNA for the parasitic trichostrongylid nematodes Trichostrongylus probolurus, Trichostrongylus rugatus and Camelostrongylus mentulatus were compared with previously published sequences for five other species within the genus Trichostrongylus. The secondary structures of the second internal transcribed spacer pre-rRNA for these nematodes were predicted using an energy minimisation method. The results indicate that a common secondary structure of the second internal transcribed spacer of these nematodes is maintained despite distinct differences in primary sequence between species. Sequence differences among Trichostrongylus species ranged from 1.3 to 7.6%, but each species differed by 22-26% in sequence when compared with C. mentulatus which belongs to a different subfamily.

Animals↗

PubMedDate not supplied

Identification of ribosome-ligand interactions using cleavage reagents.

To characterize ribosome-ligand interactions, we have used a cleavage reagent, 1,10-orthopenanthroline-Cu(II), tethered to various ligands, to cleave nearby regions of rRNA. The phenanthroline is tethered to the ligand using either an internal 4-thiouridine or a terminal thiophosphate. When Cu2+ and a reducing agent, such as mercaptopropionic acid, are present, cleavage of nearby nucleic acids occurs. The cleavage sites can be identified using primer-extension analysis. We have identified rRNA cleavage sites resulting from transcribed tRNAPhe having randomly placed phenanthroline-Cu(II), tRNAPhe with phenanthroline-Cu(II) at position 8, and a DNA oligomer complementary to positions 2655-2667 (alpha-sarcin region) with phenanthroline-Cu(II) placed at the 5' end. These results provide important new information on the structure of the rRNA within ribosomal subunits and on the proximity of rRNA neighborhoods to these bound ligands.

Base Sequence↗

PubMed1995

Organisation of the mitochondrial genome of Trichophyton rubrum. DNA sequence analysis of the ND4 gene, the ATPase subunit-6 gene, the ribosomal RNA small-subunit gene, the ND6 gene, the COXIII gene, the ATPase subunit-8 gene and six tRNA genes that correspond respectively to the tyrosine, lysine, glutamine, asparagine, isoleucine and tryptophan isoacceptors.

We present the nucleotide sequence of a 5207-bp-long region of the mitochondrial genome of the dermatophyte Trichophyton rubrum. This represents about 1/5th of the total genome and extends a previous study. From the 5' end of the present sequence, the order of genes is as follows: the end of the ND4 gene, the gene coding for subunit 6 of ATPase, the gene coding for the small ribosomal RNA (SSU rRNA), the tyrosyl tRNA gene, the ND6 gene, the COXIII gene, the ATPase 8 subunit gene and a cluster of tRNAs genes corresponding respectively to the lysine, glutamine, asparagine, isoleucine and tryptophan isoacceptors. The interesting features of this region are its compact organisation, the presence of subunit 8 of the ATPase gene and the secondary structure of SSU rRNA which is close to that of Aspergillus nidulans. On the basis of the order of the genes, which is essentially similar to that of A. nidulans, we can also assume that the LSU rRNA subunit gene should be just upstream of this sequenced region.

Adenosine Triphosphatases↗

PubMed2006

The origin and diversification of eukaryotes: problems with molecular phylogenetics and molecular clock estimation.

Determining the relationships among and divergence times for the major eukaryotic lineages remains one of the most important and controversial outstanding problems in evolutionary biology. The sequencing and phylogenetic analyses of ribosomal RNA (rRNA) genes led to the first nearly comprehensive phylogenies of eukaryotes in the late 1980s, and supported a view where cellular complexity was acquired during the divergence of extant unicellular eukaryote lineages. More recently, however, refinements in analytical methods coupled with the availability of many additional genes for phylogenetic analysis showed that much of the deep structure of early rRNA trees was artefactual. Recent phylogenetic analyses of a multiple genes and the discovery of important molecular and ultrastructural phylogenetic characters have resolved eukaryotic diversity into six major hypothetical groups. Yet relationships among these groups remain poorly understood because of saturation of sequence changes on the billion-year time-scale, possible rapid radiations of major lineages, phylogenetic artefacts and endosymbiotic or lateral gene transfer among eukaryotes. Estimating the divergence dates between the major eukaryote lineages using molecular analyses is even more difficult than phylogenetic estimation. Error in such analyses comes from a myriad of sources including: (i) calibration fossil dates, (ii) the assumed phylogenetic tree, (iii) the nucleotide or amino acid substitution model, (iv) substitution number (branch length) estimates, (v) the model of how rates of evolution change over the tree, (vi) error inherent in the time estimates for a given model and (vii) how multiple gene data are treated. By reanalysing datasets from recently published molecular clock studies, we show that when errors from these various sources are properly accounted for, the confidence intervals on inferred dates can be very large. Furthermore, estimated dates of divergence vary hugely depending on the methods used and their assumptions. Accurate dating of divergence times among the major eukaryote lineages will require a robust tree of eukaryotes, a much richer Proterozoic fossil record of microbial eukaryotes assignable to extant groups for calibration, more sophisticated relaxed molecular clock methods and many more genes sampled from the full diversity of microbial eukaryotes.

Eukaryotic Cells↗

PubMed1989

Mutations in ribosomal proteins S4 and S12 influence the higher order structure of 16 S ribosomal RNA.

We have studied the effects of protein mutations on the higher order structure of 16 S rRNA in Escherichia coli ribosomes, using a set of structure-sensitive chemical probes. Ten mutant strains were studied, which contained alterations in ribosomal proteins S4 and S12, including double mutants containing both altered S4 and S12. Two ribosomal ambiguity (ram) S4 mutant strains, four streptomycin resistant (SmR) S12 mutant strains, one streptomycin pseudodependent (SmP) S12 mutant strain, one streptomycin dependent (SmD) S12 mutant strain and two streptomycin independent (Sm1) double mutants (containing both-SmD and ram mutations) were probed and compared to an isogenic wild-type strain. In ribosomes from strains containing S4 ram mutations, nucleotides A8 and A26 become more reactive to dimethyl sulfate (DMS) at their N-1 positions. In ribosomes from strains bearing the SmD allele, A908, A909, A1413 and G1487 are significantly less reactive to chemical probes. These same effects are observed when the S4 and S12 mutations are present simultaneously in the double mutants. An interesting correlation is found between the reactivity of A908 and the miscoding potential of SmR, SmD, SmP and wild-type ribosomes; the reactivity of A908 increases as the translational error frequency of the ribosomes increases. In the case of ram ribosomes, the reactivity of A908 resembles that of wild-type, unless tRNA is bound, in which case it becomes hyper-reactive. Similarly, streptomycin has little effect on A908 in wild-type ribosomes unless tRNA is bound, in which case its reactivity increases to resemble that of ram ribosomes with bound tRNA. Finally, interaction of streptomycin with SmP and SmD ribosomes causes the reactivity of A908 to increase to near-wild-type levels. A simple model is proposed, in which the reactivity of A908 reflects the position of an equilibrium between two conformational states of the 30 S subunit, one of which is DMS-reactive, and the other DMS-unreactive. In this model, the balance between these two states would be influenced by proteins S4 and S12. Mutations in S12 generally cause a shift toward the unreactive conformer, and in the case of SmD and SmP ribosomes, this shift can be suppressed phenotypically by streptomycin, ram mutations in protein S4 cause a shift toward the reactive conformer, but only when tRNA is bound. This suggests that the opposing effects of these two classes of mutations influence the proof-reading process by somewhat different mechanisms.

Autoradiography↗

PubMed2004

Mechanisms underlying the evolution and maintenance of functionally heterogeneous 18S rRNA genes in Apicomplexans.

In many species of the protist phylum Apicomplexa, ribosomal RNA (rRNA) gene copies are structurally and functionally heterogeneous, owing to distinct requirements for rRNA-expression patterns at different developmental stages. The genomic mechanisms underlying the maintenance of this system over long-term evolutionary history are unclear. Therefore, the aim of this study was to investigate what processes underlie the long-term evolution of apicomplexan 18S genes in representative species. The results show that these genes evolve according to a birth-and-death model under strong purifying selection, thereby explaining how divergent 18S genes are generated over time while continuing to maintain their ability to produce fully functional rRNAs. In addition, it was found that Cryptosporidium parvum undergoes a rapid form of birth-and-death evolution that may facilitate host-specific adaptation, including that of type I and II strains found in humans. This represents the first case in which an rRNA gene family has been found to evolve under the birth-and-death model.

Animals↗

PubMed1988

Resistance of both the 5'- and 3'-domain of isolated Escherichia coli 23S rRNA against digestion with alpha-sarcin.

The ribonuclease alpha-sarcin exclusively cleaves the phosphodiester bond after G2661 in the 23S rRNA within 50S subunits, thus inactivating the ribosomes. The resulting alpha-fragment is 243 nucleotides long and contains the 3'-end of the 23S rRNA. The specificity is changed dramatically if isolated 23S rRNA is used as substrate. We have shown previously that 23S rRNA is digested completely except for two fragments, one of which is identical to the alpha-fragment. Here we show that the other fragment comprises the 5'-end of 23S rRNA and contains 385 nucleotides. A similar fragment was obtained when isolated 23S rRNA was digested with RNase A (specific for pyrimidines in single strands). It appears that the 5'-domain (equivalent to 5.8S rRNA of eukaryotic ribosomes) as well as the 3'-domain (equivalent to 4.5S rRNA of chloroplast ribosomes) have a compact and defined tertiary structure in isolated 23S rRNA in contrast to the rRNA region in between. Thus, alpha-sarcin is a convenient tool for detecting compact domains in isolated RNA.

Base Sequence↗

PubMed2006

Hydroxyl radical footprinting in vivo: mapping macromolecular structures with synchrotron radiation.

We used a high flux synchrotron X-ray beam to map the structure of 16S rRNA and RNase P in viable bacteria in situ. A 300 ms exposure to the X-ray beam was sufficient for optimal cleavage of the phosphodiester backbone. The in vivo footprints of the 16S rRNA in frozen cells were similar to those obtained in vitro and were consistent with the predicted accessibility of the RNA backbone to hydroxyl radical. Protection or enhanced cleavage of certain nucleotides in vivo can be explained by interactions with tRNA and perturbation of the subunit interface. Thus, short exposures to a synchrotron X-ray beam can footprint the tertiary structure and protein contacts of RNA-protein complexes with nucleotide resolution in living cells.

Base Sequence↗

PubMed2005

Mutational analysis of 16S and 23S rRNA genes of Thermus thermophilus.

Structural studies of the ribosome have benefited greatly from the use of organisms adapted to extreme environments. However, little is known about the mechanisms by which ribosomes or other ribonucleoprotein complexes have adapted to functioning under extreme conditions, and it is unclear to what degree mutant phenotypes of extremophiles will resemble those of their counterparts adapted to more moderate environments. It is conceivable that phenotypes of mutations affecting thermophilic ribosomes, for instance, will be influenced by structural adaptations specific to a thermophilic existence. This consideration is particularly important when using crystal structures of thermophilic ribosomes to interpret genetic results from nonextremophilic species. To address this issue, we have conducted a survey of spontaneously arising antibiotic-resistant mutants of the extremely thermophilic bacterium Thermus thermophilus, a species which has featured prominently in ribosome structural studies. We have accumulated over 20 single-base substitutions in T. thermophilus 16S and 23S rRNA, in the decoding site and in the peptidyltransferase active site of the ribosome. These mutations produce phenotypes that are largely identical to those of corresponding mutants of mesophilic organisms encompassing a broad phylogenetic range, suggesting that T. thermophilus may be an ideal model system for the study of ribosome structure and function.

Anti-Bacterial Agents↗

PubMed1978

Structure of the yeast ribosomes. Proteins associated with the rRNA.

Polyamines have been shown to bind to doubled stranded regions of rRNA [3]. Therefore, ribosomal proteins that can be cross linked to these molecules in the ribosomes structure must be bound to or located in the vicinity of the RNA. This technique is the first to yield results on the proteins associated with the rRNA in the eukaryotic ribosome where the lack of purified ribosomal proteins does not allow the use of direct binding studies as in bacterial systems. Proteins S7, S10, S13, S21, S22 and S27 in the small subunit and L2/3, L5, L10/12, L19/20, L22, L23, L36/37, L42 and L43' in the large subunit are labelled when cross linked to [14C]spermidine using 1,5-difluoro 2,4-dinitrobenzene and are good candidates to be RNA-binding proteins in ribosomes from Saccharomyces cerevisiae.

Molecular Weight↗

PubMed1994

Crystallography of halophilic ribosome: the isolation of an internal ribonucleoprotein complex.

Crystals of 50S ribosomal subunits from Haloarcula marismortui diffracting to 2.9 A resolution were grown. Because of their large unit cells and the extremely weak diffracting power, almost all X-ray crystallographic analysis of these crystals must be performed with intense synchrotron radiation. At ambient temperature, all ribosomal crystals decay upon the first instance of X-irradiation. To overcome this severe sensitivity, procedures for data collection at cryo temperature were developed. Under these conditions the crystals can be irradiated for periods sufficient for the collection of more than one data set from an individual crystal (days or weeks) with no observable damage. They also can be stored for months, to resume interrupted measurements. To assist the interpretation of the anticipated electron density map, a specific internal nucleoprotein complex of protein HmaL1 and a stretch of H23S rRNA was isolated from the halophilic ribosome. The fragments of the 23S rRNA protected by the protein from nuclease digestion were sequenced. Alignment of the sequences of some archaebacterial L1-specific RNA fragments to the corresponding parts of eubacterial and eukaryotic rDNAs, localized the sequence identities to two distinct regions. Chimeric complexes were reconstituted with the corresponding E. coli ribosomal components, indicating a rather high homology, despite the evolution distance. A feasible secondary structure of the rRNA stretch participating in this complex was found to be compatible with the one proposed for the corresponding part in the E. coli ribosomal RNA.

Base Sequence↗

PubMed1995

The action of pokeweed antiviral protein and ricin A-chain on mutants in the alpha-sarcin loop of Escherichia coli 23S ribosomal RNA.

The alpha-sarcin loop of Escherichia coli 23S rRNA is a universally-conserved structure involved in the binding of elongation factors Tu and G and is the site of action of the ribosome-inactivating proteins (RIPs). One such group, the N-glycosidase RIPs, act by the removal of a single adenine residue (A2660) believed to exist in a GAGA-containing tetraloop structure. The action of two RIPs, the catalytic A-chain from the heterodimeric toxic lectin ricin (RTA) and the single-chain RIP pokeweed antiviral protein (PAP), which are known to be highly homologous in tertiary structure, was determined on native ribosomes or naked 23S rRNA containing mutations designed to affect the structure of the GAGA tetraloop. One such mutant rRNA containing G2663C, which abolishes the potential tetraloop by disrupting the Watson-Crick base-pair involved in closing it, resulted in a loss of depurination by RTA, but not by PAP. A similar result was observed for mutant G2661A. The double mutant C2658G + G2663C, which restores the tetraloop-closing base-pair but in the reverse orientation, resulted in sensitivity to both PAP and RTA, as in the wild-type. Thus, the tetraloop structure is required for the action of RTA, but not of PAP, and unlike RTA, PAP does not require G at position 2661. RNA containing the G2664C mutation, which lies outside the tetraloop, served as a substrate for both PAP and RTA. The comparison of the recognition elements for PAP and RTA was made with naked (deproteinised) rRNA, because RTA does not act on E. coli ribosomes. However, PAP is active on E. coli ribosomes, and it was found that the action of PAP on ribosomes containing the above mutations paralleled exactly that on the corresponding naked rRNAs. It is concluded that the recognition elements for PAP and RTA differ and may account, at least in part, for the fact that PAP but not RTA catalyses the depurination of E. coli ribosomes.

Antiviral Agents↗

PubMed1994

Characterization of the nuclear large-subunit rRNA-encoding gene and the group-I self-splicing intron from Chlorella ellipsoidea C-87.

We found two group-I self-splicing introns in both the large subunit (LSU) and small subunit (SSU) of the nuclear rRNA-encoding genes (rDNA) of the unicellular green alga, Chlorella ellipsoidea C-87 (Ce). The primary and secondary structures of the LSU rRNA (3350 nt) and its intron (445 nt) were characterized. The intron was inserted in the conserved stem 32 of the LSU rRNA and contained all P1-P10 motifs of the group-IB intron. In vitro transcripts of the LSU rDNA containing the intron sequence displayed a strong self-splicing activity at high salt concentrations. The overall structure and splicing conditions of the LSU rRNA intron were, however, considerably different from those of the SSU rRNA intron of the same organism. These results suggest different origins and/or different evolutionary courses of these Ce introns.

Base Sequence↗

PubMed2002

Using secondary structure to identify ribosomal numts: cautionary examples from the human genome.

The identification of inadvertently sequenced mitochondrial pseudogenes (numts) is critical to any study employing mitochondrial DNA sequence data. Failure to discriminate numts correctly can confound phylogenetic reconstruction and studies of molecular evolution. This is especially problematic for ribosomal mtDNA genes. Unlike protein-coding loci, whose pseudogenes tend to accumulate diagnostic frameshift or premature stop mutations, functional ribosomal genes are not constrained to maintain a reading frame and can accumulate insertion-deletion events of varying length, particularly in nonpairing regions. Several authors have advocated using structural features of the transcribed rRNA molecule to differentiate functional mitochondrial rRNA genes from their nuclear paralogs. We explored this approach using the mitochondrial 12S rRNA gene and three known 12S numts from the human genome in the context of anthropoid phylogeny and the inferred secondary structure of primate 12S rRNA. Contrary to expectation, each of the three human numts exhibits striking concordance with secondary structure models, with little, if any, indication of their pseudogene status, and would likely escape detection based on structural criteria alone. Furthermore, we show that the unwitting inclusion of a particularly ancient (18-25 Myr old) and surprisingly cryptic human numt in a phylogenetic analysis would yield a well-supported but dramatically incorrect conclusion regarding anthropoid relationships. Though we endorse the use of secondary structure models for inferring positional homology wholeheartedly, we caution against reliance on structural criteria for the discrimination of rRNA numts, given the potential fallibility of this approach.

Base Sequence↗

PubMedDate not supplied

Recognition determinants for proteins and antibiotics within 23S rRNA.

Ribosomal RNAs fold into phylogenetically conserved secondary and tertiary structures that determine their function in protein synthesis. We have investigated Escherichia coli 23S rRNA to identify structural elements that interact with antibiotic and protein ligands. Using a combination of molecular genetic and biochemical probing techniques, we have concentrated on regions of the rRNA that are connected with specific functions. These are located in different domains within the 23S rRNA and include the ribosomal GTPase-associated center in domain II, which contains the binding sites for r-proteins L10.(L12)4 and L11 and is inhibited by interaction with the antibiotic thiostrepton. The peptidyltransferase center within domain V is inhibited by macrolide, lincosamide, and streptogramin B antibiotics, which interact with the rRNA around nucleotide A2058. Drug resistance is conferred by mutations here and by modification of A2058 by ErmE methyltransferase. ErmE recognizes a conserved motif displayed in the primary and secondary structure of the peptidyl transferase loop. Within domain VI of rRNA, the alpha-sarcin stem-loop is associated with elongation factor binding and is the target site for ribotoxins including the N-glycosidase ribosome-inactivating proteins ricin and pokeweed antiviral protein (PAP). The orientations of the 23S rRNA domains are constrained by tetiary interactions, including a pseudoknot in domain II and long-range base pairings in the center of the molecule that bring domains II and V closer together. The phenotypic effects of mutations in these regions have been investigated by expressing 23S rRNA from plasmids. Allele-specific priming sites have been introduced close to these structures in the rRNA to enable us to study the molecular events there.

Anti-Bacterial Agents↗

PubMed2003

In situ accessibility of small-subunit rRNA of members of the domains Bacteria, Archaea, and Eucarya to Cy3-labeled oligonucleotide probes.

Low accessibility of the rRNA is together with cell wall impermeability and low cellular ribosome content a frequent reason for failure of whole-cell fluorescence hybridization with fluorescently labeled oligonucleotide probes. In this study we compare accessibility data for the 16S rRNA of Escherichia coli (gamma Proteobacteria, Bacteria) with the phylogenetically distantly related organisms Pirellula sp. strain 1 (Planctomycetes, Bacteria) and Metallosphaera sedula (Crenarchaeota, Archaea) and the 18S rRNA accessibility of Saccharomyces cerevisiae (Eucarya). For a total of 537 Cy3-labeled probes, the signal intensities of hybridized cells were quantified under standardized conditions by flow cytometry. The relative probe-conferred fluorescence intensities are shown on color-coded small-subunit rRNA secondary-structure models. For Pirellula sp., most of the probes belong to class II and III (72% of the whole data set), whereas most of the probes targeting sites on M. sedula were grouped into class V and VI (46% of the whole data set). For E. coli, 45% of all probes of the data set belong to class III and IV. A consensus model for the accessibility of the small-subunit rRNA to oligonucleotide probes is proposed which uses 60 homolog target sites of the three prokaryotic 16S rRNA molecules. In general, open regions were localized around helices 13 and 14 including target positions 285 to 338, whereas helix 22 (positions 585 to 656) and the 3' half of helix 47 (positions 1320 to 1345) were generally inaccessible. Finally, the 16S rRNA consensus model was compared to data on the in situ accessibility of the 18S rRNA of S. cerevisiae.

Bacteria↗

PubMed1977

Characterization of hybrid plasmids carrying individual ribosomal ribonucleic acid transcription units of Escherichia coli.

We have screened the strains with ColE1 hybrid plasmids constructed by Clarke and Carbon (Cell 9:91-99, 1976) for the presence of ribosomal ribonucleic acid (rRNA) genes on the plasmids and identified 16 strains whose plasmids carry rRNA genes. The structures of these 16 plasmids were compared by heteroduplex analysis, and the plasmids were classified into six groups on the basis of their chromosomal origins. Homology with known transducing-phage deoxyribonucleic acids and genetic mapping have assigned locations on the Escherichia coli chromosome to three of the six groups. These are rrnB near rif at 88 min, rrnC near ilvE at 83 min, and rrnD near aroE at 71 min. A fourth group is probably rrnA at 85 min (T. Ikemura and M. Nomura, Cell, 11:779-793, 1977). We conclude that the minimum number of rRNA transcription units per haploid chromosomes is seven, that is, the six groups identified in this work plus a known operon (rrnE near metA at 89 min) that we failed to find among the hybrid plasmids. This heteroduplex analysis also suggests that there are only two kinds of rRNA operons with respect to their spacer region; three of the six rRNA operon groups studied here have one kind, whereas the remaining three have the other kind.

Bacterial Proteins↗

PubMed1982

Secondary structure of Bombyx mori and Dictyostelium discoideum 5S rRNA from S1 nuclease and cobra venom ribonuclease susceptibility, and computer assisted analysis.

The 5S rRNAs from Bombyx mori and Dictyostelium discoideum were end-labeled with [32-P] at either the 5' or 3' end and sequenced using enzymatic digestion. The secondary structure of these molecules was studied using the single-strand specific S1 nuclease and the base-pair specific cobra venom ribonuclease. Computer analysis of these results was performed and was used to generate a consensus secondary structure for each molecule. A comparison of these results with those of other workers is presented.

Animals↗