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PubMed1983

Structure and organization of rRNA operons in the region of the replication origin of the Bacillus subtilis chromosome.

Structure and organization of two complete ribosomal RNA (rRNA) gene sets, rrnO and rrnA, were determined for the first time in Bacillus subtilis. They are located at the region of the replication origin of the chromosome. Each set constitutes a single operon of: two tandem promoters - leader sequence - 16S rRNA gene - Ile-tRNA gene - Ala-tRNA gene - 23S rRNA gene - 5S rRNA gene - termination signal. The first promoter (P1) of rrnO differs from that of rrnA in sequence and function. P1 of rrnO was used very little for transcription either in vivo or in vitro while P1 was predominantly used in rrnA. A putative transcript of the entire operon was determined and constructed into a secondary structure. Analysis of in vivo transcripts by S1 mapping revealed primary processing sites at the loop and stem structure of 16S rRNA in rrnO and rrnA. A unique sequence in the leader region of rrnO can be formed into a highly complexed secondary structure and affects processing of mature 16S rRNA. The sequences of the two spacer tRNA genes are highly conserved between B. subtilis and Escherichia coli.

Bacillus subtilis↗

PubMed1992

The nucleotide sequence of the entire ribosomal DNA operon and the structure of the large subunit rRNA of Giardia muris.

The total nucleotide sequence of the rDNA of Giardia muris, an intestinal protozoan parasite of rodents, has been determined. The repeat unit is 7668 basepairs (bp) in size and consists of a spacer of 3314 bp, a small-subunit rRNA (SSU-rRNA) gene of 1429, and a large-subunit rRNA (LSU-rRNA) gene of 2698 bp. The spacer contains long direct repeats and is heterogeneous in size. The LSU-rRNA of G. muris was compared to that of the human intestinal parasite Giardia duodenalis, to the bird parasite Giardia ardeae, and to that of Escherichia coli. The LSU-rRNA has a size comparable to the 23S rRNA of E. coli but shows structural features typical for eukaryotes. Some variable regions are typically small and account for the overall smaller size of this rRNA. The structure of the G. muris LSU-rRNA is similar to that of the other Giardia rRNA, but each rRNA has characteristic features residing in a number of variable regions.

Animals↗

PubMed1981

The primary and secondary structure of yeast 26S rRNA.

We present the sequence of the 26S rRNA of the yeast Saccharomyces carlsbergensis as inferred from the gene sequence. The molecule is 3393 nucleotides long and consists of 48% G+C; 30 of the 43 methyl groups can be located in the sequence. Starting from the recently proposed structure of E. coli 23S rRNA (see ref. 25) we constructed a secondary structure model for yeast 26S rRNA. This structure is composed of 7 domains closed by long-range base pairings as n the bacterial counterpart. Most domains show considerable conservation of the overall structure; unpaired regions show extended sequence homology and the base-paired regions contain many compensating base pair changes. The extra length of the yeast molecule is due to a number of insertions in most of the domains, particularly in domain II. Domain VI, which is extremely conserved, is probably part of the ribosomal A site. alpha-Sarcin, which apparently inhibits the EF-1 dependent binding of aminoacyl-tRNA, causes a cleavage between position 3025 and 3026 in a conserved loop structure, just outside domain VI. Nearly all of the located methyl groups, like in E. coli, are present in domain II, V and VI and clustered to a certain extent mainly in regions with a strongly conserved primary structure. The only three methyl groups of 26S rRNA which are introduced relatively late during the processing are found in single stranded loops in domain VI very close to positions which have been shown in E. coli 23S rRNA to be at the interface of the ribosome.

Base Sequence↗

PubMed1993

Structural requirements of 5S rRNA for nuclear transport, 7S ribonucleoprotein particle assembly, and 60S ribosomal subunit assembly in Xenopus oocytes.

Structural requirements of 5S rRNA for nuclear transport and RNA-protein interactions have been studied by analyzing the behavior of oocyte-type 5S rRNA and of 31 different in vitro-generated mutant transcripts after microinjection into the cytoplasm of Xenopus oocytes. Experiments reveal that the sequence and secondary and/or tertiary structure requirements of 5S rRNA for nuclear transport, storage in the cytoplasm as 7S ribonucleoprotein particles, and assembly into 60S ribosomal subunits are complex and nonidentical. Elements of loops A, C, and E, helices II and V, and bulged and hinge nucleotides in the central domain of 5S rRNA carry the essential information for these functional activities. Assembly of microinjected 5S rRNA into 60S ribosomal subunits was shown to occur in the nucleus; thus, the first requirement for subunit assembly is nuclear targeting. The inhibitory effects of ATP depletion, wheat germ agglutinin, and chilling on the nuclear import of 5S rRNA indicate that it crosses the nuclear envelope through the nuclear pore complex by a pathway similar to that used by karyophilic proteins.

Animals↗

PubMed2003

Models of the primary and secondary structure for the 12S rRNA of birds: a guideline for sequence alignment.

Models of the primary and secondary structure for the 12S ribosomal RNA (rRNA) gene of birds is presented based on a comparison of 100 species. Preliminary higher-order structures were delimited following the model for vertebrates. Paired regions were refined following a complementary base-pairing criterion and compensatory mutations were considered as a further confirmation of their existence. The model shows 40 stems, 20 internal loops and 17 external loops, arranged in the typical four domains suggested for the small subunit rRNA. The higher-order structures recovered in the model were used to build a multiple sequence alignment appropriate for phylogenetic analysis. The phylogeny recovered from this alignment was compared with trees inferred from alignments assembled using different alignment parameters in the program ClustalW. The alignment based on the secondary structure is sensitive to positional covariation of stems. Nonetheless, the phylogeny recovered with this method resulted in relationships that are more congruent with non-molecular data than those inferred from alternative alignments.

Amino Acid Sequence↗

PubMed2006

Methyltransferase that modifies guanine 966 of the 16 S rRNA: functional identification and tertiary structure.

N(2)-Methylguanine 966 is located in the loop of Escherichia coli 16 S rRNA helix 31, forming a part of the P-site tRNA-binding pocket. We found yhhF to be a gene encoding for m(2)G966 specific 16 S rRNA methyltransferase. Disruption of the yhhF gene by kanamycin resistance marker leads to a loss of modification at G966. The modification could be rescued by expression of recombinant protein from the plasmid carrying the yhhF gene. Moreover, purified m(2)G966 methyltransferase, in the presence of S-adenosylomethionine (AdoMet), is able to methylate 30 S ribosomal subunits that were purified from yhhF knock-out strain in vitro. The methylation is specific for G966 base of the 16 S rRNA. The m(2)G966 methyltransferase was crystallized, and its structure has been determined and refined to 2.05A(.) The structure closely resembles RsmC rRNA methyltransferase, specific for m(2)G1207 of the 16 S rRNA. Structural comparisons and analysis of the enzyme active site suggest modes for binding AdoMet and rRNA to m(2)G966 methyltransferase. Based on the experimental data and current nomenclature the protein expressed from the yhhF gene was renamed to RsmD. A model for interaction of RsmD with ribosome has been proposed.

Escherichia coli↗

PubMed1991

The longest 18S ribosomal RNA ever known. Nucleotide sequence and presumed secondary structure of the 18S rRNA of the pea aphid, Acyrthosiphon pisum.

An EMBL4 recombinant phage which encodes one of the full length of the aphid ribosomal DNA has been isolated from the aphid genomic library. Determination of the complete nucleotide sequence of the aphid 18S rRNA gene revealed that it is 2469 bp with a G + C content of 59%. The aphid 18S rRNA gene studied here is the longest and has the highest G + C content among the 18S rRNA genes examined so far. Evidence provided by the S1 nuclease assay suggests that the aphid 18S rRNA gene examined in this study is not a pseudogene containing an insertion sequence. Based on the nucleotide sequence of the 18S rRNA gene, we constructed a presumed secondary-structure model of the aphid 18S rRNA. In the aphid 18S rRNA, the eucaryote-specific E21 and 41 region are supposed to be longer and more complex than the counterparts of other 18S rRNA.

Animals↗

PubMed2006

Mitochondrial 12S rRNA gene mutations affect RNA secondary structure and lead to variable penetrance in hearing impairment.

Mutations in the mitochondrial DNA are one of the most important causes of sensorineural hearing loss, especially in the 12S ribosomal RNA (rRNA) gene. We have analyzed the mtDNA 12S rRNA gene in a cohort of 443 families with hearing impairment, and have identified the A1555G mutation in 69 unrelated cases. A1555G is not a fully penetrant change, since only 63% of subjects with this change have developed hearing impairment. In addition, only 22% of the 183 A1555G deaf subjects were treated with aminoglycosides. Two novel nucleotide changes (T1291C and T1243C) were identified. T1243C was found in five deafness cases and one control sample. Mutation T1291C was detected in all maternally related individuals of a pedigree and in none of 95 control samples. Conservation analysis and comparison of the 12S rRNA structure with the 16S rRNA of Escherichia coli showed that the T at nucleotide 1243 and A at nucleotide 1555 are conserved positions. Prediction of RNA secondary structure showed changes in all 12S rRNA variants, the most severe being for T1291C. The reported data confirm the high prevalence of mutation A1555G in deafness cases and the major role of the 12S rRNA gene in hearing. The two novel changes reported here might have different contributions as deafness-related variants. T1291C fulfills the criteria of a disease-causing change. As in the case of mutation A1555G, the underlying phenotype of T1291C is not homogeneous for all family members, providing evidence for the implication of environmental and/or additional genetic factors.

Adult↗

PubMed1991

Involvement of "hinge" nucleotides of Xenopus laevis 5 S rRNA in the RNA structural organization and in the binding of transcription factor TFIIIA.

Nucleotides in the bifurcation region of the 5 S rRNA, the junction of the three helical domains, play a central role in determining the coaxial stacking interactions and tertiary structure of the RNA. We have used site-directed mutagenesis of Xenopus laevis oocyte 5 S rRNA to make all possible nucleotide substitutions at three positions in loop A (10, 11 and 13) and at the G66.U109 base-pair at the beginning of helix V. Certain double point mutations were constructed to ascertain the relationship between loop A nucleotides and the G.U base-pair. The importance of the size of the bifurcation region was tested by the creation of a single nucleotide deletion mutant and two single nucleotide insertion mutants. The effects of these mutations on the structure and function of the 5 S rRNA were determined by solution structure probing of approximately half of the mutants with chemical reagents, and by measuring the relative binding affinity of each mutant for transcription factor TFIIIA. Proposed structural rearrangements in the bifurcation region were tested by using a graphic modeling method combining stereochemical constraints and chemical reactivity data. From this work, several insights were obtained into the general problem of helix stacking and RNA folding at complex bifurcation regions. None of the mutations caused an alteration of the coaxial stacking of helix V on helix II proposed for the wild-type 5 S rRNA. However, the formation of a Watson-Crick pair between nucleotide 13 of loop A and nucleotide 66 at the top of helix V does cause a destabilization of the proximal part of this helix. Also, nucleotide 109 at the top of helix V will preferentially pair with nucleotide 10 of loop A rather than nucleotide 66 when both possibilities are provided, without affecting the stability of helix V, even though the G.U pair is disrupted. The effects of these mutations on TFIIIA binding indicate that the bifurcation region is critical for protein recognition. One important feature of the relationship between 5 S rRNA structure and TFIIIA recognition resulting from this study was the observation that any mutation that constrains the bifurcation loop results in a reduced affinity of the RNA for TFIIIA, unless it is compensated for by an increased flexibility elsewhere.

Animals↗

PubMed1990

Secondary structures of Tetrahymena thermophila rRNA IVS sequence involved in its self-splicing reactions: a new computer analysis.

The secondary structures of Tetrahymena thermophila rRNA IVS sequence involved in the self-splicing reactions, are theoretically investigated with a refined computer method previously proposed, able to select a set of the deepest free energy RNA secondary structures under constraints of model hypotheses and experimental evidences. The secondary structures obtained are characterized by the close proximity of self-reactions sites and account for double mutations experiments, and differential digestion data.

Animals↗

PubMed1994

The antibiotics micrococcin and thiostrepton interact directly with 23S rRNA nucleotides 1067A and 1095A.

The antibiotics thiostrepton and micrococcin bind to the GTPase region in domain II of 23S rRNA, and inhibit ribosomal A-site associated reactions. When bound to the ribosome, these antibiotics alter the accessibility of nucleotides 1067A and 1095A towards chemical reagents. Plasmid-coded Escherichia coli 23S rRNAs with single mutations at positions 1067 or 1095 were expressed in vivo. Mutant ribosomes are functional in protein synthesis, although those with transversion mutations function less effectively. Antibiotics were bound under conditions where wild-type and mutant ribosomes compete in the same reaction for drug molecules; binding was analysed by allele-specific footprinting. Transversion mutations at 1067 reduce thiostrepton binding more than 1000-fold. The 1067G substitution gives a more modest decrease in thiostrepton binding. The changes at 1095 slightly, but significantly, lower the affinity of ribosomes for thiostrepton, again with the G mutation having the smallest effect. Micrococcin binding to ribosomes is reduced to a far greater extent than thiostrepton by all the 1067 and 1095 mutations. Extrapolating these results to growing cells, mutation of nucleotide 1067A confers resistance towards micrococcin and thiostrepton, while substitutions at 1095A confer micrococcin resistance, and increase tolerance towards thiostrepton. These data support an rRNA tertiary structure model in which 1067A and 1095A lie in close proximity, and are key components in the drug binding site. None of the mutations alters either the higher order rRNA structure or the binding of r-proteins. We therefore conclude that thiostrepton and micrococcin interact directly with 1067A and 1095A.

Anti-Bacterial Agents↗

PubMed1985

Electron microscopy of secondary structure in partially denatured precursor and mature Escherichia coli 16 S and 23 S rRNA.

The secondary structure of 16 S and 23 s rRNA sequences in 30 S preribosomal RNA of Escherichia coli was analyzed by electron microscopy after partial denaturation and compared to mature 16 S and 23 S rRNA examined under the same conditions. The sequences in the pre-rRNA notably lack the specific loops that dominate the 5'-terminal regions of mature 16 S and 23 S rRNA. In other respects, the sizes and locations of loops in the 23 S rRNA sequence are qualitatively very similar in mature and pre-rRNA. Eleven of 12 loops outside of the 5'-terminal domain correspond, with the most frequent features in the 3'-half of the molecule. In contrast, the sizes and locations of loops in the 16 S rRNA sequence differ between precursor and mature forms. In the pre-rRNA, instead of the 370-nucleotide 5'-terminal loop of mature rRNA, some 1000-nucleotide terminal loops are observed. The pre-rRNA also shows a frequent 610-nucleotide central loop and a large 1240-nucleotide loop not seen in the mature rRNA. Also, in the 3'-region of the sequence, the largest loops in pre-rRNA are 120 nucleotides shorter than in mature rRNA. We suggest that the structure of pre-rRNA may promote some alternate conformational features, and that these could be important during ribosome formation or function.

Escherichia coli↗

PubMed1975

The primary structure of the 5s rRNA binding protein L25 of Escherichia coli ribosomes.

The primary structure of protein L25 from the large subunit of Escherichia coli ribosomes was determined by isolation and analysis of peptides obtained after cleavage of the protein with trypsin, thermolysin and Staphylococcus protease as well as by Edman degradation of the intact protein and of a CNBr peptide. The complete amino acid sequence is shown in Fig. 4. There are sequence homologies within protein L25 (Table 6) as well as between protein L25 and other ribosomal proteins (Table 5).

Amino Acid Sequence↗

PubMed2002

The accuracy of ribosomal RNA comparative structure models.

The determination of the 16S and 23S rRNA secondary structure models was initiated shortly after the first complete 16S and 23S rRNA sequences were determined in the late 1970s. The structures that are common to all 16S rRNAs and all 23S rRNAs were determined using comparative methods from the analysis of thousands of rRNA sequences. Twenty-plus years later, the 16S and 23S rRNA comparative structure models have been evaluated against the recently determined high-resolution crystal structures of the 30S and 50S ribosomal subunits. Nearly all of the predicted covariation-based base pairs, including the regular base pairs and helices, and the irregular base pairs and tertiary interactions, were present in the 30S and 50S crystal structures.

Crystallography, X-Ray↗

PubMed2003

[Characterization of 5S rRNA gene sequence and secondary structure in gymnosperms].

In higher plants the primary and the secondary structures of 5S ribosomal RNA gene are considered highly conservative. Little is known about the 5S rRNA gene structure, organization and variation in gyimnosperms. In this study we analyzed sequence and structure variation of 5S rRNA gene in Pinus through cloning and sequencing multiple copies of 5S rDNA repeats from individual trees of five pines, P. bungeana, P. tabulaeformis, P. yunnanensis, P. massoniana and P. densata. Pinus bungeana is from the subgenus Strobus while the other four are from the subgenus Pinus (diploxylon pines). Our results revealed variations in both primary and secondary structure among copies of 5S rDNA within individual genomes and between species. 5S rRNA gene in Pinus is 120 bp long in most of the 122 clones we sequenced except for one or two deletions in three clones. Among these clones 50 unique sequences were identified and they were shared by different pine species. Our sequences were compared to 13 sequences each representing a different gymnosperm species, and to six sequences representing both angiosperm monocots and dicots. Average sequence similarity was 97.1% among Pinus species and 94.3% between Pinus and other gymnosperms. Between gymnosperms and angiosperms the sequence similarity decreased to 88.1%. Similar to other molecular data, significant sequence divergence was found between the two Pinus subgenera. The 5S gene tree (neighbor-joining tree) grouped the four diploxylon pines together and separated them distinctly from P. bungeana. Comparison of sequence divergence within individuals and between species suggested that concerted evolution has been very weak especially after the divergence of the four diploxylon pines. The phylogenetic information contained in the 5S rRNA gene is limited due to its shorter length and the difficulties in identifying orthologous and paralogous copies of rDNA multigene family further complicate its phylogenetic application. Pinus densata is a diploid hybrid between P. tabulaeformis and P. yunnanensis. Its 5S rDNA composition is consistent with its hybrid origin. 5S rRNA of all gymnosperms published so far could be folded into a general secondary structure. Variation in this secondary structure was detected among species. About 55% of the 120 bp nucleotide positions was variable, in which 68% was on stem regions. Nevertheless, the positions at the end of the stems and those adjacent to loops are conserved. Their stability directly determines the size of the loops. Some mutations such as compensatory base-pair substitutions, and G-U pairing could be regarded as mechanisms for maintaining a stable secondary structure. The loops of the secondary structure are also relatively conserved. It seems that stable helices are necessary for the function of the gene. The conserved nucleotides in the loops are probably involved in the interaction with proteins and/or RNAs or with other nucleotide in the formation of the tertiary structure. However, unlike other reports, Loop E was found quite mutable among pines. These variations together with those on stems might be caused by the presence of pseudogenes among our clones. A preliminary evaluation indicates that only seven of 50 unique sequences are potentially functional genes.

Base Sequence↗

PubMed2005

Homology modeling revealed more than 20,000 rRNA internal transcribed spacer 2 (ITS2) secondary structures.

Structural genomics meets phylogenetics and vice versa: Knowing rRNA secondary structures is a prerequisite for constructing rRNA alignments for inferring phylogenies, and inferring phylogenies is a precondition to understand the evolution of such rRNA secondary structures. Here, both scientific worlds go together. The rRNA internal transcribed spacer 2 (ITS2) region is a widely used phylogenetic marker. Because of its high variability at the sequence level, correct alignments have to take into account structural information. In this study, we examine the extent of the conservation in structure. We present (1) the homology modeled secondary structure of more than 20,000 ITS2 covering about 14,000 species; (2) a computational approach for homology modeling of rRNA structures, which additionally can be applied to other RNA families; and (3) a database providing about 25,000 ITS2 sequences with their associated secondary structures, a refined ITS2 specific general time reversible (GTR) substitution model, and a scoring matrix, available at http://its2.bioapps.biozentrum.uni-wuerzburg.de.

Animals↗

PubMed1982

Primary and secondary structure of 5.8S rRNA from the silkgland of Bombyx mori.

Nucleotide sequence of 5.8S rRNA of the silkworm, Bombyx mori has been determined by gel sequencing methods. The 5.8S rRNA was the longest so far reported, with the 5'-terminal sequence several nucleotides longer than those of the other organisms. Upon constructing the secondary structure in accordance with the "burp gun" model (12), the Bombyx 5.8S rRNA formed a wide-open "muzzle" due to several unpaired bases at the ends. The overall structure also appeared less stable with less G . C pairs and more unpaired bases than that of the HeLa 5.8S rRNA. These structural features may be essential for those 5.8S rRNAs which interact with 28S rRNAs containing the hidden break to form a stable complex.

Animals↗

PubMedDate not supplied

[Secondary structure of pre-rRNA 41S spacers and its possible role in determining the processing sites].

The mechanism of pre-rRNA processing is still unknown. In this paper we discuss a possible role of the secondary structure of the internal transcribed spacer region (ITS-1 and ITS-2) in processing of 41S pre-rRNA. Potential possibilities for double strand structure formation in ITS-1 and ITS-2 and its rearrangement in the course of processing of 41S pre-rRNA with involvement of U3 RNA were analyzed for a number of species. We have concluded that this rearrangement is the necessary stage in 41S pre-rRNA processing when every functionally designated area (for mature 18S, 28S and 5,8S rRNAs, for ITS-1, ITS-2 and 21S pre-rRNA) has to be organized into an individual domain. In these strongly structured domains 5'- and 3'-ends are spatially brought together whereas processing sites are localized between these compactised areas.

Animals↗