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PubMed1981

Is wheat mitochondrial 5S ribosomal RNA prokaryotic in nature?

Küntzel et al. (1981) (Nucleic Acids Res. 9, 1451-1461) recently concluded that the sequence of wheat mitochondrial 5S rRNA is significantly more related to prokaryotic than to eukaryotic 5S rRNA sequences, and displays an especially high affinity to that of the thermophilic Gram-negative bacterium, Thermus aquaticus. However, the sequence on which this conclusion was based, although attributed to us, differs in several places from the one determined by us. We show here that the correct sequence (Spencer, D.F., Bonen, L. and Gray, M.W. (1981) Biochemistry, in press) does not support the conclusions of Küntzel et al. about potential secondary structure in wheat mitochondrial 5S rRNA and its phylogenetic significance. We further show that when the wheat mitochondrial 5S rRNA sequence is matched against published alignments for E. coli, T. aquaticus, and wheat cytosol 5S rRNAs, the mitochondrial sequence shows no greater homology to the T. aquaticus sequence than to the E. coli sequence, and only slightly more homology to these two sequences than to wheat cytosol 5S rRNA. This analysis confirms our original view (Biochemistry, in press) that wheat mitochondrial 5S rRNA is neither obviously prokaryotic nor eukaryotic in nature, but shows characteristics of both classes of 5S rRNA, as well as some unique features.

Base Sequence↗

PubMed1988

Isolation of cDNA clones coding for mitochondrial 16S ribosomal RNA from the crustacean Artemia.

cDNA clones coding for Artemia mitochondrial 16S ribosomal RNA (rRNA) have been isolated. The clones cover from nucleotide 650 of the RNA molecule to its 3' end. The comparison of Artemia sequence with both vertebrate and invertebrate mitochondrial 16S rRNA sequences has shown the existence of regions of high similarity between them. A model for the secondary structure of the 3' half of Artemia mitochondrial 16S rRNA is proposed. The size of the rRNA molecule has been estimated at 1.35 kb. Despite the similarity of the Artemia gene to insect rRNA in size, sequence and secondary structure, the G + C content of the Artemia gene (42%) is closer to that of mammals than to the insect genes. The number of mitochondria in Artemia has been estimated at 1500 per diploid genome in the cyst and 4000 in the nauplius. In contrast, the amount of mt 16S rRNA is constant at all stages of Artemia development.

Animals↗

PubMed1999

Seven novel methylation guide small nucleolar RNAs are processed from a common polycistronic transcript by Rat1p and RNase III in yeast.

Through a computer search of the genome of the yeast Saccharomyces cerevisiae, the coding sequences of seven different box C/D antisense small nucleolar RNAs (snoRNAs) with the structural hallmarks of guides for rRNA ribose methylation have been detected clustered over a 1.4-kb tract in an inter-open reading frame region of chromosome XIII. The corresponding snoRNAs have been positively identified in yeast cells. Disruption of the nonessential snoRNA gene cluster specifically suppressed the seven cognate rRNA ribose methylations but did not result in any growth delay under the conditions of yeast culture tested. The seven snoRNAs are processed from a common polycistronic transcript synthesized from an independent promoter, similar to some plant snoRNAs but in marked contrast with their vertebrate functional homologues processed from pre-mRNA introns containing a single snoRNA. Processing of the polycistronic precursor requires nucleases also involved in rRNA processing, i.e., Rnt1p and Rat1p. After disruption of the RNT1 gene, the yeast ortholog of bacterial RNase III, production of the seven mature snoRNAs was abolished, while the polycistronic snoRNA precursor accumulated. In cells lacking functional Rat1p, an exonuclease involved in the processing of both pre-rRNA and intron-encoded snoRNAs, several processing intermediates of the polycistronic precursor accumulated. This allowed for the mapping in the precursor of the presumptive Rnt1p endonucleolytic cuts which provide entry sites for subsequent exonucleolytic trimming of the pre-snoRNAs. In line with known properties of double-stranded RNA-specific RNase III, pairs of Rnt1p cuts map next to each other on opposite strands of long double-helical stems in the secondary structure predicted for the polycistronic snoRNA precursor.

Base Sequence↗

PubMed2000

Mutations at position A960 of E. coli 23 S ribosomal RNA influence the structure of 5 S ribosomal RNA and the peptidyltransferase region of 23 S ribosomal RNA.

The proximity of loop D of 5 S rRNA to two regions of 23 S rRNA, domain II involved in translocation and domain V involved in peptide bond formation, is known from previous cross-linking experiments. Here, we have used site-directed mutagenesis and chemical probing to further define these contacts and possible sites of communication between 5 S and 23 S rRNA. Three different mutants were constructed at position A960, a highly conserved nucleotide in domain II previously crosslinked to 5 S rRNA, and the mutant rRNAs were expressed from plasmids as homogeneous populations of ribosomes in Escherichia coli deficient in all seven chromosomal copies of the rRNA operon. Mutations A960U, A960G and, particularly, A960C caused structural rearrangements in the loop D of 5 S rRNA and in the peptidyltransferase region of domain V, as well as in the 960 loop itself. These observations support the proposal that loop D of 5 S rRNA participates in signal transmission between the ribosome centers responsible for peptide bond formation and translocation.

Aldehydes↗

PubMed1988

Mutagenesis at the mRNA decoding site in the 16S ribosomal RNA using the specialized ribosome system in Escherichia coli.

In the specialized ribosome system, a distinct pool of mutated ribosomes is dedicated to the translation of one particular mRNA species. This was accomplished by altering the Shine-Dalgarno sequence on the mRNA and its complementary anti-Shine-Dalgarno sequence on the plasmid-borne 16S rRNA gene. Here, using the specialized ribosome system, we were able to introduce mutations in key regions of the 16S rRNA and could study their effect on translation in vivo. The C1400 region has been implicated to play a role in the actual mRNA decoding process. Several ribosomal mutations were introduced in this region. We showed that substitution of the evolutionary highly conserved C1400 residue by a G- or an A-residue inhibits ribosomal activity by 80% and 50% respectively, whereas, a C to a U change at this conserved position does not affect overall ribosomal activity. The adjacent stem structure (1410-1490) was also examined. Disruption of the stem by replacing either one of the arms of this stem, with a different sequence, inhibits ribosomal activity by approximately 80%. A small but significant restoration of translation could be achieved by recreating a complementary stem with a different sequence. We found that full reversion of activity could be obtained when such mutated ribosomes were made spectinomycin resistant by introducing a C to A substitution at position 1192 which is located far away in the secondary structure map of the 16S rRNA molecule. Based on these results we conclude that some, but not all, of the nucleotides in the conserved C1400 region play a key role in translation.(ABSTRACT TRUNCATED AT 250 WORDS)

Escherichia coli↗

PubMed1999

Putative secondary structures of unusually long strepsipteran SSU rRNAs and its phylogenetic implications.

We constructed the putative secondary structures of the small subunit rRNAs (SSU rRNA) from three strepsipteran insects. The primary sequences of the strepsipteran SSU rRNAs are unusually long due to unique and long insertions. In spite of these insertions, the basic shapes of their secondary structures are well maintained as shown in those of other eukaryotes, because these insertions appear mainly in the variable regions. The secondary structures for the V1, V3, V5, V8, and V9 regions are well conserved, even though the primary structures of V1, V5, and V8 regions are quite variable. However, the predicted secondary structures for the V2, V4, and V7 regions are quite different from those of other insects. In the V4 and V7 regions, helices specific to the Strepsiptera exist. These helices have not been reported in other organisms so far. Similarly, four eukaryotic specific helices (E8-1, E10-2, E23-4 and E45-1) not reported in insects exist in the V2, V4, and V8 regions. These helices are formed by the inserted sequences. The secondary structures of the expanded segments of the strepsipteran SSU rRNA were applied to infer the phylogenetic position of Strepsiptera, one of the most enigmatic problems in insect phylogeny. Only the secondary structure of the V7 region showed the weak Strepsiptera/Diptera sister-group relationship.

Animals↗

PubMed1988

Evolution of the secondary structures and compensatory mutations of the ribosomal RNAs of Drosophila melanogaster.

This paper examines the effects of DNA sequence evolution on RNA secondary structures and compensatory mutations. Models of the secondary structures of Drosophila melanogaster 18S ribosomal RNA (rRNA) and of the complex between 2S, 5.8S, and 28S rRNAs have been drawn on the basis of comparative and energetic criteria. The overall AU richness of the D. melanogaster rRNAs allows the resolution of some ambiguities in the structures of both large rRNAs. Comparison of the sequence of expansion segment V2 in D. melanogaster 18S rRNA with the same region in three other Drosophila species and the tsetse fly (Glossina morsitans morsitans) allows us to distinguish between two models for the secondary structure of this region. The secondary structures of the expansion segments of D. melanogaster 28S rRNA conform to a general pattern for all eukaryotes, despite having highly divergent sequences between D. melanogaster and vertebrates. The 70 novel compensatory mutations identified in the 28S rRNA show a strong (70%) bias toward A-U base pairs, suggesting that a process of biased mutation and/or biased fixation of A and T point mutations or AT-rich slippage-generated motifs has occurred during the evolution of D. melanogaster rDNA. This process has not occurred throughout the D. melanogaster genome. The processes by which compensatory pairs of mutations are generated and spread are discussed, and a model is suggested by which a second mutation is more likely to occur in a unit with a first mutation as such a unit begins to spread through the family and concomitantly through the population. Alternatively, mechanisms of proofreading in stem-loop structures at the DNA level, or between RNA and DNA, might be involved. The apparent tolerance of noncompensatory mutations in some stems which are otherwise strongly supported by comparative criteria within D. melanogaster 28S rRNA must be borne in mind when compensatory mutations are used as a criterion in secondary-structure modeling. Noncompensatory mutation may extend to the production of unstable structures where a stem is stabilized by RNA-protein or additional RNA-RNA interactions in the mature ribosome. Of motifs suggested to be involved in rRNA processing, one (CGAAAG) is strongly overrepresented in the 28S rRNA sequence. The data are discussed both in the context of the forces involved with the evolution of multigene families and in the context of molecular coevolution in the rDNA family in particular.

Animals↗

PubMed1995

U24, a novel intron-encoded small nucleolar RNA with two 12 nt long, phylogenetically conserved complementarities to 28S rRNA.

Following computer searches of sequence banks, we have positively identified a novel intronic snoRNA, U24, encoded in the ribosomal protein L7a gene in humans and chicken. Like previously reported intronic snoRNAs, U24 is devoid of a 5'-trimethyl-cap. U24 is immunoprecipitated by an antifibrillarin antibody and displays an exclusively nucleolar localization by fluorescence microscopy after in situ hybridization with antisense oligonucleotides. In vertebrates, U24 is a 76 nt long conserved RNA which is metabolically stable, present at approximately 14,000 molecules per human HeLa cell. U24 exhibits a 5'-3' terminal stem-box C-box D structure, typical for several snoRNAs, and contains two 12 nt long conserved sequences complementary to 28S rRNA. It is, therefore, strikingly related to U14, U20 and U21 snoRNAs which also possess long sequences complementary to conserved sequences of mature 18S or 28S rRNAs. In 28S rRNA the two tracts complementary to U24 are adjacent to each other, they involve several methylated nucleotides and are surprisingly close, within the rRNA secondary structure, to complementarities to snoRNAs U18 and U21. Identification of the yeast Saccharomyces cerevisiae U24 gene directly confirms the outstanding conservation of the complementarity to 28S rRNA during evolution, suggesting a key role of U24 pairing to pre-rRNA during ribosome biogenesis, possible in the control of pre-rRNA folding. Yeast S.cerevisiae U24 is also intron-encoded but not in the same host-gene as in humans or chicken.

Animals↗

PubMed1995

Evolution and structural conservation of the control region of insect mitochondrial DNA.

The control regions of mitochondrial DNA of two insects, Schistocerca gregaria and Chorthippus parallelus, have been isolated and sequenced. Their sizes are 752 bp and 1,512 bp, respectively, with the presence of a tandem repeat in C. parallelus. (The sequences of the two repeats are highly conserved, having a homology of 97.5%.) Comparison of their nucleotide sequences revealed the presence of several conserved sequence blocks dispersed through the whole control region, showing a different evolutionary pattern of this region in these insects as compared to that in Drosophila. A highly conserved secondary structure, located in the 3' region near the small rRNA gene, has been identified. Sequences immediately flanking this hairpin structure rather than the sequences of this structure themselves are conserved between S. gregaria/C. parallelus and Drosophila, having a sequence consensus of "TATA" at 5' and "GAA(A)T" at 3'. The motif "G(A)nT" is also present in the 3' flanking sequences of mammalian, amphibian, and fish mitochondrial L-strand replication origins and a potential plant mitochondrial second-strand-replication origin, indicating its universal conservation and functional importance related to replication origins. The stem-and-loop structure in S. gregaria/C. parallelus appears to be closely related to that found in Drosophila despite occupying a different position, and may be potentially associated with a second-strand-replication origin. This in turn suggests that such a secondary structure might be widely conserved across invertebrates while their location in the control region may be variable. We have looked for such a conserved structure in the control regions of two other insects, G. firmus and A. mellifera, whose DNA sequences have been published, and their possible presence is discussed. Mitochondrial control regions characterized to date in five different insect taxa (Drosophila, G. firmus, A. mellifera, S. gregaria, and C. parallelus) may be classed into two distinct groups having different evolutionary patterns. It is observed that tandem repetition of regions containing a probable replication origin occurred in some species from disjunct lineages in both groups, which would be the result of convergent evolution. We also discuss the possibility of a mechanism of "parahomologous recombination by unequal crossing-over" in mitochondria, which can explain the generation of such tandemly repeated sequences (especially the first critical repetition) in the control region of mtDNA, and also their convergent evolution in disjunct biological lineages during evolution.

Animals↗

PubMed2005

Molecular identification of lizard by RAPD & FINS of mitochondrial 16s rRNA gene.

In the present study, we identified the structure-less skeleton suspected to be of house lizard present in jaggery, consumption of which caused mass food poisoning using, RAPD (Random Amplification of Polymorphic DNA) with random primers and FINS (Forensically Informative Nucleotide Sequencing) with mitochondrial 16s rRNA gene. The NJ tree dendogram based on distance calculated from RAPD bands clearly identified the structure-less as Calotes versicolor (Garden Lizard). In FINS analysis of the mitochondrial 16s rRNA gene the NJ tree based on Kimura-2-parameter distance matrices clearly reveal that the unknown sample clustered with Agmidae family and closest to Calotes versicolor (Garden Lizard) with 100% bootstrap support, whereas all other species belong to Gekkonida family form a single distinct cluster including Hemidactylus fluviviridis (House Lizard). This is the first successful typing of mitochondrial 16s rRNA with FINS approach to identify the biological origin of a structure-less skeleton. Our analysis also sustained successful identification of unknown samples using RAPD method with optimized conditions in a laboratory setup with low resources.

Animals↗

PubMed2000

A map of protein-rRNA distribution in the 70 S Escherichia coli ribosome.

Neutron scattering exploits the enormous scattering difference between protons and deuterons. A set of 42 x-ray and neutron solution scattering curves from hybrid Escherichia coli ribosomes was obtained, where the proteins and rRNA moieties in the subunits were either protonated or deuterated in all possible combinations. This extensive data set is analyzed using a novel method. The volume defined by the cryoelectron microscopic model of Frank and co-workers (Frank, J., Zhu, J., Penczek, P., Li, Y. H., Srivastava, S., Verschoor, A., Radermacher, M., Grassucci, R., Lata, R. K., and Agrawal, R. K. (1995) Nature 376, 441-444) is divided into 7890 densely packed spheres of radius 0.5 nm. Simulated annealing is employed to assign each sphere to solvent, protein, or rRNA moieties to simultaneously fit all scattering curves. Twelve independent reconstructions starting from random approximations yielded reproducible results. The resulting model at a resolution of 3 nm represents the volumes occupied by rRNA and protein moieties at 95% probability threshold and displays 15 and 20 protein subvolumes in the 30 S and 50 S, respectively, connected by rRNA. 17 proteins with known atomic structure can be tentatively positioned into the protein subvolumes within the ribosome in agreement with the results from other methods. The protein-rRNA map enlarges the basis for the models of the rRNA folding and can further help to localize proteins in high-resolution crystallographic density maps.

Bacterial Proteins↗

PubMed1996

A misfolded form of 5S rRNA is complexed with the Ro and La autoantigens.

In both vertebrate and invertebrate cells, the 60-kDa Ro autoantigen is bound to small cytoplasmic RNAs known as Y RNAs. In Xenopus oocytes, the 60-kDa Ro protein is also complexed with a class of 5S rRNA precursors that contain internal mutations. Because these 5S rRNA precursors are processed inefficiently and degraded eventually, the Ro protein may function in a quality control pathway for 5S rRNA biosynthesis. We have investigated the sequence and secondary structure determinants in the mutant 5S rRNAs that confer binding by the 60-kDa Ro protein. The mutant 5S rRNAs fold to form an alternative helix that is required for recognition by the 60-kDa Ro protein. Mutations that disrupt the alternative helix eliminate Ro protein binding, whereas compensatory changes that restore the helix are bound efficiently by the Ro protein. When the structure of the mutant RNA was probed using dimethylsulfate and oligonucleotide-directed RNase H cleavage, the results were consistent with the formation of the alternative structure. The La protein, which is also complexed with the mutant 5S rRNA precursors, protects similar sequences from nuclease digestion as does the 60-kDa Ro protein. Thus, the binding sites for these two proteins are either nearby on the RNA, or the two proteins may be complexed through protein-protein interactions. When the human Ro protein is expressed in the yeast Saccharomyces cerevisiae, the protein binds wild-type 5S rRNA precursors, suggesting that a population of wild-type precursors also folds into the alternative structure.

Animals↗

PubMed1989

Thermus thermophilus 16S rRNA is transcribed from an isolated transcription unit.

A cloned 16S rRNA gene from the extreme thermophilic eubacterium Thermus thermophilus HB8 was used to characterize the in vivo expression of the 16S rRNA genes in this organism by nuclease S1 mapping. The gene represents an isolated transcription unit encoding solely 16S rRNA. Under exponential growth conditions, transcription was initiated at a single promoter, which represents the structural equivalent of Escherichia coli rrn P2 promoters. The promoter-leader region was very similar to the E. coli rrn P2 promoter-leader segment that is responsible for antitermination. The T. thermophilus leader region was approximately 85 nucleotides shorter than its E. coli P2 counterpart. Potential processing intermediates were correlated with a proposed secondary structure of T. thermophilus pre-16S rRNA.

Base Sequence↗

PubMed1982

Fragmentary 5S rRNA gene in the human mitochondrial genome.

The human mitochondrial genome contains a 23-nucleotide sequence that is homologous to a part of the 5S rRNA's of bacteria. This homology, the structure of the likely transcript, and the location of the sequence relative to the mitochondrial rRNA genes suggest that the sequence represents a fragmentary 5S rRNA gene.

Base Sequence↗

PubMed2005

Structural implications of novel diversity in eucaryal RNase P RNA.

Previous eucaryotic RNase P RNA secondary structural models have been based on limited diversity, representing only two of the approximately 30 phylogenetic kingdoms of the domain Eucarya. To elucidate a more generally applicable structure, we used biochemical, bioinformatic, and molecular approaches to obtain RNase P RNA sequences from diverse organisms including representatives of six additional kingdoms of eucaryotes. Novel sequences were from acanthamoeba (Acathamoeba castellanii, Balamuthia mandrillaris, Filamoeba nolandi), animals (Caenorhabditis elegans, Drosophila melanogaster), alveolates (Theileria annulata, Babesia bovis), conosids (Dictyostelium discoideum, Physarum polycephalum), trichomonads (Trichomonas vaginalis), microsporidia (Encephalitozoon cuniculi), and diplomonads (Giardia intestinalis). An improved alignment of eucaryal RNase P RNA sequences was assembled and used for statistical and comparative structural analysis. The analysis identifies a conserved core structure of eucaryal RNase P RNA that has been maintained throughout evolution and indicates that covariation in size occurs between some structural elements of the RNA. Eucaryal RNase P RNA contains regions of highly variable length and structure reminiscent of expansion segments found in rRNA. The eucaryal RNA has been remodeled through evolution as a simplified version of the structure found in bacterial and archaeal RNase P RNAs.

Acanthamoeba castellanii↗

PubMed1994

Chromatin structure and transcriptional activity around the replication forks arrested at the 3' end of the yeast rRNA genes.

Replication intermediates containing forks arrested at the replication fork barrier near the 3' end of the yeast rRNA genes were analyzed at the chromatin level by using in vivo psoralen cross-linking as a probe for chromatin structure. These specific intermediates were purified from preparative two-dimensional agarose gels, and the extent of cross-linking in the different portions of the branched molecules was examined by electron microscopy and by using a psoralen gel retardation assay. The unreplicated section corresponding to the rRNA coding region upstream of the arrested forks appeared mostly heavily cross-linked, characteristic of transcriptionally active rRNA genes devoid of nucleosomes, whereas the replicated daughter strands representing newly synthesized spacer sequences showed a nucleosomal organization typical for bulk chromatin. The failure to detect replication forks arrested at the 3' end of inactive rRNA gene copies and the fact that most DNA encoding rRNA (rDNA) is replicated in the same direction as transcription suggest that replication forks seldom originate from origins of replication located immediately downstream of inactive genes.

Chromatin↗

PubMed2004

Protein phylogenies robustly resolve the deep-level relationships within Euglenozoa.

The deepest-level relationships amongst Euglenozoa remain poorly resolved, despite a rich history of morphological examination and numerous molecular phylogenetic studies of small subunit ribosomal RNA (SSU rRNA) data. We address this question using two nuclear-encoded proteins, the cytosolic isoforms of heat shock protein 90 (hsp90) and heat shock protein 70 (hsp70). For both proteins we examined sequences from the three primary groups within Euglenozoa (euglenids, diplonemids, and kinetoplastids), and from their close relatives, Heterolobosea. Maximum likelihood (ML) and ML distance analyses of these proteins support a close relationship between diplonemids and kinetoplastids to the exclusion of the euglenid Euglena gracilis. In hsp90 and combined protein analyses bootstrap support is very strong and alternative topologies are generally rejected by 'approximately unbiased' (AU) tests. This result is consistent with recent molecular biological and morphological data, but contradicts early structural accounts and many SSU rRNA analyses that favour a closer relationship between diplonemids and euglenids. However, a re-examination of an important SSU rRNA data set highlights the instability of the inferences from this marker. The protein analyses also suggest that bodonids are paraphyletic, with trypanosomatids grouping with 'clade 2' and 'clade 3' bodonids to the exclusion of 'clade 1' bodonids.

Animals↗

PubMed2006

Domain organization and crystal structure of the catalytic domain of E.coli RluF, a pseudouridine synthase that acts on 23S rRNA.

Pseudouridine synthases catalyze the isomerization of uridine to pseudouridine (Psi) in rRNA and tRNA. The pseudouridine synthase RluF from Escherichia coli (E.C. 4.2.1.70) modifies U2604 in 23S rRNA, and belongs to a large family of pseudouridine synthases present in all kingdoms of life. Here we report the domain architecture and crystal structure of the catalytic domain of E.coli RluF at 2.6A resolution. Limited proteolysis, mass spectrometry and N-terminal sequencing indicate that RluF has a distinct domain architecture, with the catalytic domain flanked at the N and C termini by additional domains connected to it by flexible linkers. The structure of the catalytic domain of RluF is similar to those of RsuA and TruB. RluF is a member of the RsuA sequence family of Psi-synthases, along with RluB and RluE. Structural comparison of RluF with its closest structural homologues, RsuA and TruB, suggests possible functional roles for the N-terminal and C-terminal domains of RluF.

Amino Acid Sequence↗