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PubMed1999

The two faces of the Escherichia coli 23 S rRNA sarcin/ricin domain: the structure at 1.11 A resolution.

The sarcin/ricin domain of 23 S - 28 S ribosomal RNA is essential for protein synthesis because it forms a critical part of the binding site for elongation factors. A crystal structure of an RNA of 27 nucleotides that mimics the domain in Escherichia coli 23 S rRNA was determined at 1.11 A resolution. The domain folds into a hairpin distorted by four non-canonical base-pairs and one base triple. The fold is stabilized by cross-strand and intra-stand stacking; no intramolecular stabilizing metal ions are observed. This is the first structure to reveal in great detail the geometry and the hydration of two common motifs that are conserved in this rRNA domain, a GAGA tetraloop and a G-bulged cross-strand A stack. Differences in the region connecting these motifs to the stem in the E. coli and in the rat sarcin/ricin domains may contribute to the species-specific binding of elongation factors. Correlation of nucleotide protection data with the structure indicates that the domain has two surfaces. One surface is accessible, lies primarily in the major groove, and is likely to bind the elongation factors. The second lies primarily in the minor groove, and is likely to be buried in the ribosome. This minor groove surface includes the Watson-Crick faces of the cytosine bases in the unusual A2654.C2666 and U2653.C2667 water-mediated base-pairs.

Animals↗

PubMed1986

Identification of the binding site on 5S rRNA for the transcription factor IIIA: proposed structure of a common binding site on 5S rRNA and on the gene.

Transcription factor IIIA interacts specifically with an internal control region of Xenopus 5S ribosomal RNA genes and is also a component, along with 5S rRNA, of a 7S ribonucleoprotein particle present in previtellogenic oocytes. We have determined the region of the 5S rRNA in the 7S ribonucleoprotein complex that is protected by the transcription factor from digestion with the ribonuclease alpha-sarcin. The binding site for factor IIIA extends from nucleotide 64 through nucleotide 116; the protected region includes two CCUGG helices separated by 11 nucleotides. The same helices occur in the factor IIIA binding site in the 5S rRNA gene and may constitute a common structural feature recognized by the protein in the gene and in the gene product.

Animals↗

PubMed1996

Structural analysis of two plant 5S rRNA species and fragments thereof by lead-induced hydrolysis.

The structure of plant 5S rRNA species from lupin and wheat germ as well as the structure of two RNA fragments that represent domains beta and gamma of lupin 5S rRNA have been probed by Pb(II)-induced hydrolysis. The lead digestion patterns of 5S rRNA species show that the secondary and tertiary structures of the molecules are very similar. The data suggests that two potential base pairs at the bottom of helix E are destabilized and this causes an enlargement of the hairpin loop e. On the other hand, nucleotides from loop c seem to be involved in the formation of some kind of higher order structure. A comparison of the distribution of cleavages induced in RNA fragments to those in the corresponding regions of the entire 5S rRNA shows that under conditions applied in our studies the structural domains beta and gamma are not involved in formation of any tertiary interaction within 5S rRNA structure.

Base Sequence↗

PubMed1984

Paramecium mitochondrial genes. I. Small subunit rRNA gene sequence and microevolution.

The sequences of the small subunit mitochondrial rRNA genes from two divergent species of Paramecium (primaurelia and tetraurelia) were determined. The gene lies near the center of the linear mitochondrial genome, on the same strand as are all other currently identified genes. The sequences generally resemble their counterparts found in cytoplasmic, procaryotic, and other mitochondrial sources. The rDNA gene boundaries were located by nuclease S1 protection. Small subunit rDNA spans about 1680 nucleotides, including an extraneous 83-base pair sequence very near the 3' end which is unique to Paramecium mitochondria. This "insert" occurs at the apex of the highly variable in length penultimate helix, according to proposed models for small subunit rRNA secondary structure. A discontinuity occurs in isolated rRNA near the start of the insert, resulting in a stable 13 S RNA species and a small segment containing the remaining 3' portion of the gene. The overall rRNA gene sequence was 94% conserved between the two species, and the nucleotide differences consisted of 53% transitions, 37% transversions, and 9% insertions plus deletions. These substitutions were somewhat clustered, and the two most divergent regions coincided with the gene boundaries. The sequence was aligned with Escherichia coli 16 S rRNA for direct comparison of sequence and structure.

Animals↗

PubMed2002

Structure of the 16S rRNA pseudouridine synthase RsuA bound to uracil and UMP.

In Escherichia coli, the pseudouridine synthase RsuA catalyzes formation of pseudouridine (psi) at position 516 in 16S rRNA during assembly of the 30S ribosomal subunit. We have determined the crystal structure of RsuA bound to uracil at 2.0 A resolution and to uridine 5'-monophosphate (UMP) at 2.65 A resolution. RsuA consists of an N-terminal domain connected by an extended linker to the central and C-terminal domains. Uracil and UMP bind in a cleft between the central and C-terminal domains near the catalytic residue Asp 102. The N-terminal domain shows structural similarity to the ribosomal protein S4. Despite only 15% amino acid identity, the other two domains are structurally similar to those of the tRNA-specific psi-synthase TruA, including the position of the catalytic Asp. Our results suggest that all four families of pseudouridine synthases share the same fold of their catalytic domain(s) and uracil-binding site.

Amino Acid Sequence↗

PubMed2004

Evaluation of the suitability of free-energy minimization using nearest-neighbor energy parameters for RNA secondary structure prediction.

BACKGROUND: A detailed understanding of an RNA's correct secondary and tertiary structure is crucial to understanding its function and mechanism in the cell. Free energy minimization with energy parameters based on the nearest-neighbor model and comparative analysis are the primary methods for predicting an RNA's secondary structure from its sequence. Version 3.1 of Mfold has been available since 1999. This version contains an expanded sequence dependence of energy parameters and the ability to incorporate coaxial stacking into free energy calculations. We test Mfold 3.1 by performing the largest and most phylogenetically diverse comparison of rRNA and tRNA structures predicted by comparative analysis and Mfold, and we use the results of our tests on 16S and 23S rRNA sequences to assess the improvement between Mfold 2.3 and Mfold 3.1. RESULTS: The average prediction accuracy for a 16S or 23S rRNA sequence with Mfold 3.1 is 41%, while the prediction accuracies for the majority of 16S and 23S rRNA structures tested are between 20% and 60%, with some having less than 20% prediction accuracy. The average prediction accuracy was 71% for 5S rRNA and 69% for tRNA. The majority of the 5S rRNA and tRNA sequences have prediction accuracies greater than 60%. The prediction accuracy of 16S rRNA base-pairs decreases exponentially as the number of nucleotides intervening between the 5' and 3' halves of the base-pair increases. CONCLUSION: Our analysis indicates that the current set of nearest-neighbor energy parameters in conjunction with the Mfold folding algorithm are unable to consistently and reliably predict an RNA's correct secondary structure. For 16S or 23S rRNA structure prediction, Mfold 3.1 offers little improvement over Mfold 2.3. However, the nearest-neighbor energy parameters do work well for shorter RNA sequences such as tRNA or 5S rRNA, or for larger rRNAs when the contact distance between the base-pairs is less than 100 nucleotides.

Base Sequence↗

PubMed1996

Nucleotide sequence and presumed secondary structure of the 28S rRNA of pea aphid: implication for diversification of insect rRNA.

Determination of the entire nucleotide sequence of the aphid 28S ribosomal RNA gene (28S rDNA) revealed that it is 4,147 bp in length with a G + C content of 60.3%. Based on the nucleotide sequence, we constructed a presumed secondary-structure model of the aphid 28S rRNA which indicated that the aphid 28S rRNA is characterized by the length and high G + C content of its variable regions. The G + C content of the aphid's variable regions was much higher than that of the entire sequence of the 28S rRNA, which formed a striking contrast to those of Drosophila with the G + C content much lower than the entire 28S molecule. In this respect, the aphid 28S rRNA somewhat resembled those of vertebrates. This is the third report of a complete large-subunit rRNA sequence from an arthropod, and the first 28S rRNA sequence for a nondipterous insect.

Animals↗

PubMed1989

DNA sequence and secondary structures of the large subunit rRNA coding regions and its two class I introns of mitochondrial DNA from Podospora anserina.

DNA sequence analysis has shown that the gene coding for the mitochondrial (mt) large subunit ribosomal RNA (rRNA) from Podospora anserina is interrupted by two class I introns. The coding region for the large subunit rRNA itself is 3715 bp and the two introns are 1544 (r1) and 2404 (r2) bp in length. Secondary structure models for the large subunit rRNA were constructed and compared with the equivalent structure from Escherichia coli 23S rRNA. The two structures were remarkably similar despite an 800-base difference in length. The additional bases in the P. anserina rRNA appear to be mostly in unstructured regions in the 3' part of the RNA. Secondary structure models for the two introns show striking similarities with each other as well as with the intron models from the equivalent introns in Saccharomyces cerevisiae, Neurospora crassa, and Aspergillus nidulans. The long open reading frames in each intron are different from each other, however, and the nucleotide sequence similarity diverges as it proceeds away from the core structure. Each intron is located within regions of the large subunit rRNA gene that are highly conserved in both sequence and structure. Computer analysis showed that the open reading frame for intron r1 contained a common maturase-like polypeptide. The open reading frames of intron r2 appeared to be chimeric, displaying high sequence similarity with the open reading frames in the r1 and ATPase 6 introns of N. crassa.

Ascomycota↗

PubMed1986

Rapid chemical probing of conformation in 16 S ribosomal RNA and 30 S ribosomal subunits using primer extension.

We have investigated in detail the higher-order structure of 16 S ribosomal RNA, both in its naked form and in 30 S ribosomal subunits. Each base in the 16 S rRNA chain has been probed using kethoxal (which reacts with guanine at N1 and N2), dimethylsulfate (which reacts with adenine at N1 and cytosine at N3) and 1-cyclohexyl-3-(2-morpholinoethyl)-carbodiimide metho-p-toluenesulfonate (which reacts with uracil at N3 and guanine at N1). The sites of reaction were identified by primer extension with reverse transcriptase using synthetic oligodeoxynucleotide primers. These results provide a detailed and rigorous experimental test of a model for 16 S rRNA secondary structure, which was derived mainly from comparative sequence analysis. Our data also provide information relevant to tertiary and quaternary structure of 16 S rRNA. Data obtained with naked 16 S rRNA show reasonably close agreement with the proposed model, and data obtained with 30 S subunits show nearly complete agreement. Apart from an apparent overall "tightening" of the structure (in which many weakly reactive bases become unreactive), assembly of the proteins with 16 S rRNA to form 30 S subunits brings about numerous local structural rearrangements, resulting in specific enhancements as well as protections. In many instances, the ribosomal proteins appear to "tune" the 16 S rRNA structure to bring it into accordance with the phylogenetically predicted model, even though the RNA on its own often seems to prefer a different structure in certain regions of the molecule. Extensive protection of conserved, unpaired adenines upon formation of 30 S subunits suggests that they play a special role in the assembly process, possibly providing signals for protein recognition.

Aldehydes↗

PubMed1990

Primary and secondary structure of the 25S rRNA from the dimorphic fungus Mucor racemosus.

A 9.76 Kb ribosomal DNA repeat unit from the nuclear genome of the dimorphic fungus Mucor racemosus (Zygomycetes) was identified using a hybridization probe from the yeast Saccharomyces cerevisiae (Ascomycetes). This material was cloned in Escherichia coli plasmids as four overlapping pieces and mapped with respect to cleavage sites for 12 restriction endonucleases. The nucleotide sequence of the complete 25S rRNA gene and flanking regions was determined. The 5' and 3' ends of the structural gene were identified by comparison with the published sequence for the S. cerevisiae gene. The Mucor gene was found to possess 3469 bp and have a GC content of 42.8%. It was compared with the homologous gene from several other eukaryotes and found to be most similar to that from Saccharomyces. A potential secondary structure of the putative RNA transcript consistent with the structures proposed for the E. coli and Saccharomyces molecules was constructed by computer modelling.

Base Sequence↗

PubMed2024

Diversity of ribosomes at the level of rRNA variation associated with human health and disease.

With hundreds of copies of rDNA, it is unknown whether they possess sequence variations that form different types of ribosomes. Here, we developed an algorithm for long-read variant calling, termed RGA, which revealed that variations in human rDNA loci are predominantly insertion-deletion (indel) variants. We developed full-length rRNA sequencing (RIBO-RT) and in situ sequencing (SWITCH-seq), which showed that translating ribosomes possess variation in rRNA. Over 1,000 variants are lowly expressed. However, tens of variants are abundant and form distinct rRNA subtypes with different structures near indels as revealed by long-read rRNA structure probing coupled to dimethyl sulfate sequencing. rRNA subtypes show differential expression in endoderm/ectoderm-derived tissues, and in cancer, low-abundance rRNA variants can become highly expressed. Together, this study identifies the diversity of ribosomes at the level of rRNA variants, their chromosomal location, and unique structure as well as the association of ribosome variation with tissue-specific biology and cancer.

Humans↗

PubMed1981

Studies on the secondary structure of 5.8 S rRNA from a thermophile, Thermomyces lanuginosus.

The nucleotide sequence of ribosomal 5.8 S RNA from a thermophilic fungus, Thermomyces lanuginosus, was determined to be (formula: see text). The secondary structure was probed under a variety of ionic conditions using limited pancreatic and T1 ribonuclease digestion and rapid gel sequencing techniques. The results generally supported the "burp gun" model previously proposed for all 5.8 S rRNAs (Nazar, R. N., Sitz, T. O., and Busch, H. (1975) J. Biol. Chem. 250, 8591-8597) and were inconsistent with a recently suggested "cloverleaf" configuration (Luoma, G. A., and Marshall, A. G. (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 4901-4905). Theoretical considerations of the overall stability also appear to favor the former estimate. As previously observed with other 5.8 S RNAs the sequence of T. lanuginosus RNA is strikingly homologous with other species; it differs in only 13 positions from that of yeast. When compared to yeast, the differences appear not to contribute to the stability of the secondary structure but probably lead to a more stable 5.8 S-25 S rRNA interaction in the large ribosomal subunit. A general comparison of T. lanuginosus 5.8 S RNA with all known 5.8 S RNA sequences indicates that, although modified nucleotides differ significantly between species, they are always located in four highly conserved regions of the 5.8 S molecule, probably contributing to the unique character of these very essential sequences.

Base Sequence↗

PubMed1987

Multiple spacer sequences in the nuclear large subunit ribosomal RNA gene of Crithidia fasciculata.

In Crithidia fasciculata, a trypanosomatid protozoan, the nuclear-encoded ;28S' rRNA is multiply fragmented, comprising two large (c and d) and four small (e, f, g and j) RNA species. We have determined that the coding sequences for these RNAs (and that of the 5.8S rRNA, species i) are separated from one another by spacer sequences ranging in size from 31 to 416 bp. Coding and spacer sequences are presumably co-transcribed, with excision of the latter during post-transcriptional processing generating a highly fragmented large subunit (LSU) rRNA. Secondary structure modelling indicates that the C. fasciculata LSU rRNA complex (seven segments, including 5.8S rRNA) is held together in part by long-range intermolecular base pairing interactions that are characteristic of intramolecular interactions in the covalently continuous LSU (23S) rRNA of Escherichia coli. At least one functionally critical region (encompassing the alpha-sarcin cleavage site) is contained in a small RNA species (f) rather than in one of the two large RNAs. Within a proposed secondary structure model of C. fasciculata LSU rRNA, discontinuities between the different segments (created by spacer excision) map to regions that are highly variable in structure in covalently continuous LSU rRNAs. We suggest that ;rRNA genes in pieces' and discontinuous rRNAs may represent an evolutionarily ancient pattern.

Journal Article↗

PubMed1994

A conserved secondary structural motif in 23S rRNA defines the site of interaction of amicetin, a universal inhibitor of peptide bond formation.

The binding site and probable site of action have been determined for the universal antibiotic amicetin which inhibits peptide bond formation. Evidence from in vivo mutants, site-directed mutations and chemical footprinting all implicate a highly conserved motif in the secondary structure of the 23S-like rRNA close to the central circle of domain V. We infer that this motif lies at, or close to, the catalytic site in the peptidyl transfer centre. The binding site of amicetin is the first of a group of functionally related hexose-cytosine inhibitors to be localized on the ribosome.

Anti-Bacterial Agents↗

PubMed1986

The importance of individual nucleotides for the structure and function of rRNA molecules in E. coli. A mutagenesis study.

Methods of in vitro mutagenesis were employed to determine the importance of individual nucleotides within the ribosomal RNAs for the structure and function of E. coli ribosomes. A series of defined nucleotides in the genes for the 5 S and 16 S RNA were altered by transition and transversion mutations using either oligonucleotide-directed or bisulfite-catalyzed mutation procedures. Plasmids harbouring the mutated rRNA genes were expressed and the ribosomes containing such altered RNAs were investigated for impairments in RNA-protein interaction assembly and mRNA-coded tRNA binding.

Base Sequence↗

PubMed1982

Sequences of three molluscan 5 S ribosomal RNAs confirm the validity of a dynamic secondary structure model.

The collection of known 5 S rRNA primary structures is enriched with the sequences from three mollusca, the snails Helix pomatia and Arion rufus, and the mussel Mytilus edulis. The three sequences can be fitted in a five-helix secondary structure model previously shown (De Wachter et al. (1982) Biochimie 64, 311-329) to apply to all 5 S RNAs regardless of their origin. One of the helices in this model can undergo a bulge-internal loop transition. Within the metazoan kingdom, the dimensions of each helix and loop are rigidly conserved, except for one helix which can comprise either 6 or 7 base pairs.

Animals↗

PubMed1996

5S rRNA sugar-phosphate backbone protection in complexes with specific ribosomal proteins.

5S ribosomal RNA forms stable specific complexes with ribosomal proteins L18, L25 and L5. In this work, interaction of phosphate residues of E. coli 5S rRNA within 5S rRNA-protein complexes has been studied. For this purpose 5S rRNA with statistically distributed phosphorothioate residues has been used for complex formation and the accessibility of phosphorothioates to iodine cleavage in the complex and in the free state has been studied. In free 5S rRNA, the phosphate residue at A73 was partially protected, probably due to being involved in the organization of the spatial structure of 5S rRNA. This protection is stronger in the complex with three proteins when the 5S rRNA structure is stabilized. In the 5S rRNA-L18 complex only two phosphate groups, G7 and A34, were protected. L25 in a complex with 5S rRNA protects large numbers of phosphorothioate groups concentrating in two clusters, indicating the possibility of two binding sites for this protein on 5S rRNA. The protection pattern differs from that for individual proteins because of the possible rearrangement of the structure.

Base Sequence↗

PubMed1994

Comparative structure and genomic organization of the discontinuous mitochondrial ribosomal RNA genes of Chlamydomonas eugametos and Chlamydomonas reinhardtii.

We report that the mitochondrial ribosomal RNAs (rRNAs) of Chlamydomonas eugametos are discontinuously encoded in separate gene pieces that are scrambled in order and interspersed with protein coding genes. Individual transcripts of these mitochondrial rRNA gene pieces have the potential to form standard rRNA secondary structures through intermolecular base-pairing and they all have termini that are confined to previously defined variable rRNA domains. The C. eugametos and the previously described Chlamydomonas reinhardtii mitochondrial DNAs, therefore, share the unusual feature of highly fragmented and extensively rearranged rRNA coding regions, which contrasts with the conventional mitochondrial rRNA gene structure of land plants and other green algae. Although many of the sites of mitochondrial rRNA discontinuity are in corresponding variable regions in the two Chlamydomonas species, several variable rRNA regions are interrupted in one species but not the other and the 5' to 3' order of the C. eugametos and C. reinhardtii gene pieces is very different. Based on these results, we conclude that the last common ancestor of C. eugametos and C. reinhardtii had discontinuous mitochondrial rRNA genes and that processes responsible for the further division and scrambling of these coding regions have continued since the divergence of C. eugametos and C. reinhardtii. The presence of four group I introns within the C. eugametos mitochondrial rRNA gene pieces leads us to favour recombination rather than reverse-transcription as the mechanism giving rise to the scrambled arrangement of rRNA genes in Chlamydomonas mitochondria.

Animals↗