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PubMed2004

Crystal structure of KsgA, a universally conserved rRNA adenine dimethyltransferase in Escherichia coli.

The bacterial enzyme KsgA catalyzes the transfer of a total of four methyl groups from S-adenosyl-l-methionine (S-AdoMet) to two adjacent adenosine bases in 16S rRNA. This enzyme and the resulting modified adenosine bases appear to be conserved in all species of eubacteria, eukaryotes, and archaebacteria, and in eukaryotic organelles. Bacterial resistance to the aminoglycoside antibiotic kasugamycin involves inactivation of KsgA and resulting loss of the dimethylations, with modest consequences to the overall fitness of the organism. In contrast, the yeast ortholog, Dim1, is essential. In yeast, and presumably in other eukaryotes, the enzyme performs a vital role in pre-rRNA processing in addition to its methylating activity. Another ortholog has been discovered recently, h-mtTFB in human mitochondria, which has a second function; this enzyme is a nuclear-encoded mitochondrial transcription factor. The KsgA enzymes are homologous to another family of RNA methyltransferases, the Erm enzymes, which methylate a single adenosine base in 23S rRNA and confer resistance to the MLS-B group of antibiotics. Despite their sequence similarity, the two enzyme families have strikingly different levels of regulation that remain to be elucidated. We have crystallized KsgA from Escherichia coli and solved its structure to a resolution of 2.1A. The structure bears a strong similarity to the crystal structure of ErmC' from Bacillus stearothermophilus and a lesser similarity to sc-mtTFB, the Saccharomyces cerevisiae version of h-mtTFB. Comparison of the three crystal structures and further study of the KsgA protein will provide insight into this interesting group of enzymes.

Amino Acid Sequence↗

PubMed2006

16S rRNA gene analyses of bacterial community structures in the soils of evergreen broad-leaved forests in south-west China.

Bacterial community structure was studied in humus and mineral soils of evergreen broad-leaved forests in Ailaoshan and Xishuangbanna, representing subtropical and tropical ecosystems, respectively, in south-west China using sequence analysis and terminal restriction fragment length polymorphism (T-RFLP) analysis of 16S rRNA genes. Clone sequences affiliated to Acidobacteria were retrieved as the predominant bacterial phylum in both forest soils, followed by those affiliated to members of the Proteobacteria, Planctomycete and Verrucomicrobia. Despite higher floristic richness at the Xishuangbanna forest than at the Ailaoshan forest, soil at Xishuangbanna harbored a distinctly high relative abundance of Acidobacteria-affiliated sequences (80% of the total clones), which led to a lower overall bacterial diversity than at Ailaoshan. Bacterial communities in humus and mineral soils of the two forests appeared to be well differentiated, based on 16S rRNA gene phylogeny, and correlations were found between the bacterial T-RFLP community patterns and the organic carbon and nutrient contents of the soil samples. The data reveal that Acidobacteria dominate soil bacterial communities in the evergreen broad-leaved forests studied here and suggest that bacterial diversity may be influenced by soil carbon and nutrient levels, but is not related to floristic richness along the climatic gradient from subtropical to tropical forests in south-west China.

Bacteria↗

PubMed1991

A conserved core structure in the 18-25S rRNA intergenic region from tobacco, Nicotiana rustica.

To identify conserved and functionally important features in the intergenic sequences of ribosomal DNAs, the nucleotide sequence of the 18-25S rRNA intergene region in tobacco rDNA was determined and compared to that of other higher plants. Unlike previous comparisons of more diverse organisms, sufficient sequence homology is retained in the higher plants to examine the evolutionary changes which make these regions diverse. Estimates of the secondary structure permit the identification of a 'core-like' structure which appears to maintain the processed sites in close proximity and can be identified in the more divergent sequences.

Base Sequence↗

PubMed2000

[Cloning and sequence analysis of SSU rRNA gene of cutaneous leishmaniasis pathogen from Xinjiang of China].

OBJECTIVE: By sequencing of SSU rRNA gene cloning from Xinjiang cutaneous leishmaniasis pathogen (XJCLP) to provide evidence for identification of the pathogen. METHODS: By PCR assay with primers R222 and R333, the specific fragment had been produced from SSU rRNA gene of XJCLP, L. infantum, L. tropica and cloned into pGEM-T Easy vector. The clones were sequenced by the Sanger dideoxy-mediated chain termination method, analysis of SSU rRNA gene sequences from XJCLP, L. tropica, L. infantum with DNASIS. RESULTS: Sequence analysis showed that the specific fragment of SSU rRNA gene from XJCLP, L. infantum, L. tropica, were all 394 bp in length. There were 391 bases identical and three point mutations between the sequences of XJCLP and L. tropica, the similarity being 99.2%; 390 bases identical and three point mutations and one insertion/deletion between the sequences of XJCLP and L. infantum, the similarity being 99.0%. One insertion/deletion between the sequences of L. tropica and L. infantum, the similarity being 99.7%. The primary and secondary structures of SSU rRNA gene from XJCLP differed from those of L. infantum and L. tropica. A retrieval from GenBank confirmed that these 394 bp sequence are new gene sequences. CONCLUSION: The primary and secondary structures of SSU rRNA gene from XJCLP, L. infantum, L. tropica were different. 394 bp sequence from SSU rRNA gene of XJCLP is a new gene sequence.

Animals↗

PubMed1997

Organization and nucleotide sequence analysis of the ribosomal gene set (rrnB) from Streptomyces lividans.

Streptomyces lividans (Sl) contains six ribosomal RNA (rRNA) gene sets, rrnA-F (Suzuki, Y., Ono, Y., Nagata, A. and Yamada, T. (1988) Molecular cloning and characterization of an rRNA operon in Streptomyces lividans TK21. J. Bacteriol. 170, 1631-1636). We have cloned the rrnB gene cluster. Physical mapping revealed that rrnB gene set is located on a 290 kb Asel fragment in the 11 to 12 o'clock region of the S. coelicolor A3(2) chromosome. The complete nucleotide (nt) sequence of Sl 23S rRNA has been determined. The structural gene of the Sl 23S rRNA codes for the 3108 nt RNA chain. The G+C content of the 23S rRNA is 57.3 mol%. The length of the spacer region between the 23S and 5S genes is 99 bp. Analysis of the sequences between the 16S and 23S genes and downstream of the 5S rRNA gene failed to identify any tRNA-like sequences. A secondary structure model of Sl 23 rRNA is proposed, based on the earlier published model of Gutell and Fox (Nucleic Acids Res. 16 (1988) 175-269).

Base Sequence↗

PubMed1984

Probing the conformation of 18S rRNA in yeast 40S ribosomal subunits with kethoxal.

Yeast 40S ribosomal subunits have been reacted with kethoxal to probe the conformation of 18S rRNA. Over 130 oligonucleotides were isolated by diagonal electrophoresis and sequenced, allowing identification of 48 kethoxal-reactive sites in the 18S rRNA chain. These results generally support a secondary structure model for 18S rRNA derived from comparative sequence analysis. Significant reactivity at positions 1436 and 1439, in a region shown to be base paired by comparative analysis, lends support to the earlier suggestion [Chapman, N.M., & Noller, H.F. (1977) J. Mol. Biol 109, 131-149] that part of the 3'-major domain of 16S-like rRNAs may undergo a biologically significant conformational rearrangement. Modification of positions in 18S rRNA analogous to those previously found for Escherichia coli 16S rRNA argues for extensive structural homology between 30S and 40S ribosomal subunits, particularly in regions thought to be directly involved in translation.

Aldehydes↗

PubMed2002

Distribution of rRNA introns in the three-dimensional structure of the ribosome.

More than 1200 introns have been documented at over 150 unique sites in the small and large subunit ribosomal RNA genes (as of February 2002). Nearly all of these introns are assigned to one of four main types: group I, group II, archaeal and spliceosomal. This sequence information has been organized into a relational database that is accessible through the Comparative RNA Web Site (http://www.rna.icmb.utexas.edu/) While the rRNA introns are distributed across the entire tree of life, the majority of introns occur within a few phylogenetic groups. We analyzed the distributions of rRNA introns within the three-dimensional structures of the 30S and 50S ribosomes. Most sites in rRNA genes that contain introns contain only one type of intron. While the intron insertion sites occur at many different coordinates, the majority are clustered near conserved residues that form tRNA binding sites and the subunit interface. Contrary to our expectations, many of these positions are not accessible to solvent in the mature ribosome. The correlation between the frequency of intron insertions and proximity of the insertion site to functionally important residues suggests an association between intron evolution and rRNA function.

Introns↗

PubMed2000

Structure of free Thermus flavus 5 S rRNA at 1.3 nm resolution from synchrotron X-ray solution scattering.

The shape of free Thermus flavus 5 S rRNA in solution at 1.3 nm resolution is restored from synchrotron x-ray scattering data using an ab initio simulated annealing algorithm. The free 5 S rRNA is a bent elongated molecule displaying a compact central region and two projecting arms, similar to those of the tRNA. The atomic models of the 5 S rRNA domains A-D-E and B-C in the form of elongated helices can be well accommodated within the shape, yielding a tentative model of the structure of the free 5 S rRNA in solution. Its comparison with the recent protein-RNA map in the ribosome (Svergun, D. I., and Nierhaus, K. H. (2000) J. Biol. Chem. 275, 14432-14439) indicates that the 5 S rRNA becomes essentially more compact upon complex formation with specific ribosomal proteins. A conceivable conformational change involves rotation of the B-C domain toward the A-D-E domain. The model of free 5 S rRNA displays no interactions between domains E and C, but such interactions are possible in the bound molecule.

Algorithms↗

PubMed1996

Differential sensitivity of 16S rRNA targeted oligonucleotide probes used for fluorescence in situ hybridization is a result of ribosomal higher order structure.

The use of 16S rRNA targeted gene probes for the direct analysis of microbial communities has revolutionized the field of microbial ecology, yet a comprehensive approach for the design of such probes does not exist. The development of 16S rRNA targeted oligonucleotide probes for use with fluorescence in situ hybridization (FISH) procedures has been especially difficult as a result of the complex nature of the rRNA target molecule. In this study a systematic comparison of 16S rRNA targeted oligonucleotide gene probes was conducted to determine if target location influences the hybridization efficiency of oligonucleotide probes when used with in situ hybridization protocols for the detection of whole microbial cells. Five unique universal 12-mer oligonucleotide sequences, located at different regions of the 16S rRNA molecule, were identified by a computer-aided sequence analysis of over 1000 partial and complete 16S rRNA sequences. The complements of these oligomeric sequences were chemically synthesized for use as probes and end labeled with either [gamma-32P]ATP or the fluorescent molecule tetramethylrhodamine-5/-6. Hybridization sensitivity for each of the probes was determined by hybridization to heat-denatured RNA immobilized on blots or to formaldehyde fixed whole cells. All of the probes hybridized with equal efficiency to denatured RNA. However, the probes exhibited a wide range of sensitivity (from none to very strong) when hybridized with whole cells using a previously developed FISH procedure. Differential hybridization efficiencies against whole cells could not be attributed to cell wall type, since the relative probe efficiency was preserved when either Gram-negative or -positive cells were used. These studies represent one of the first attempts to systematically define criteria for 16S rRNA targeted probe design for use against whole cells and establish target site location as a critical parameter in probe design.

Cell Wall↗

PubMed1990

Effect of mutations in domain 2 on the structural organization of oocyte 5 S rRNA from Xenopus laevis.

In order to test and refine the molecular model of Xenopus laevis 5 S rRNA proposed in a previous work, we have synthesized, by site-directed mutagenesis and in vitro transcription, four mutants in the internal loop B and in the hairpin loop C of domain 2. The conformations of these mutant 5 S rRNAs have been tested using a variety of enzymatic and chemical structure-specific probes and computer modeling. The mutations induce conformational changes restricted to the mutated regions. Our results demonstrate unambiguously that the three helical domains of the Y-shaped structure are independent and that loop C possesses an intrinsic conformation, which is not involved in any tertiary long-range interaction. They point to the crucial role of invariant nucleotides in maintaining the intrinsic conformation of the loop and to the effect of sequence on the stability of loop regions.

Animals↗

PubMedDate not supplied

[Comparison of the conformation of RNA from phage MS2 and 16S rRNA. Accessibility to nucleases S1 and SV specific to secondary structure and thermal stability].

The hydrolysis of E. coli 16S rRNA by nucleases specific to the secondary structure elements (S1 and SV), the melting of the RNA after partial hydrolysis by nuclease S1 and the electrophoretic mobility of hydrolysis products after denaturation-renaturation of RNA were studied. It was shown that the sensitivity of 16S rRNA to nuclease S1 depends on Zn or Mg ions concentration. The melting curves after partial hydrolysis by nuclease S1 were characterized by a decrease of the hyperchromic effect (by approximately 15%) and by a increase of Tm (by 3 degrees). After RNA denaturation followed by slow or fast renaturation the electrophoretic patterns of the hydrolysis products were not changed, as in the case of phage MS2 RNA. It was supposed, that the rRNA molecule has a stable "nucleus" (or "nuclei"), which is organized as an intramolecular association of parallelly oriented double-stranded fragments of this RNA. Previously, such a mode of the spatial organization was proposed by us for phage MS2 RNA.

Coliphages↗

PubMed2006

A CAPS test allowing a rapid distinction of Penicillium expansum among fungal species collected on grape berries, inferred from the sequence and secondary structure of the mitochondrial SSU-rRNA.

Penicillium expansum is a fungal species highly damageable for the postharvest conservation of numerous fruits. In vineyards, this fungus is sometimes isolated from grape berries where its presence may lead to the production of geosmin, a powerful earthy odorant, which can impair grapes and wines aromas. However, the discrimination of P. expansum from related fungi is difficult because it is based on ambiguous phenotypic characters and/or expensive and time-consuming molecular tests. In this context, the complete sequences and secondary structures of Penicillium expansum and Penicillium thomii mitochondrial SSU-rRNAs were achieved and compared with those of two other phylogenetically related Ascomycota: Penicillium chrysogenum and Emericella nidulans. The comparison has shown a high conservation in size and sequence of the core and of the variable domains (more than 80% of nt identity) of the four SSU-rRNAs, arguing for a close phylogenetic relationship between these four species of the Trichocomaceae family. Large (from 10 to 18 nt) inserted/deleted (indel) sequences were evidenced in the V1, V5 and V6 variable domains. The size variations (10 to 18 nt) of the V1 indel sequence allowed the distinction of the four species; the V5 indel (15 nt) was specifically recovered in E. nidulans; the V6 indel (16 nt), shared by the three Penicillium species, was lacking in E. nidulans. A couple of conserved primers (UI/R2) were defined to generate a PCR product containing the V1 to V5 variable domains. This product contained the two regions of the four SSU-rRNAs showing the highest rates of nt substitutions, namely the V2 variable domain and, surprisingly, a helix (H17) of the core. The H17 sequence was shown to specifically possess in P. expansum a recognition site for the ClaI restriction endonuclease. Hence, this enzyme generates a digestion pattern of the PCR product with two bands (350 bp+500 bp), specific to P. expansum and easily separable by agarose gel electrophoresis. This leads to a CAPS test usable for P. expansum discrimination among grape berries isolated filamentous fungi. The CAPS test was validated by a comparative analysis involving 29 strains belonging to 17 species currently isolated from grape berries in the Bordeaux vineyards.

Base Sequence↗

PubMedDate not supplied

[Primary structure of the 5.8S gene of rRNA and internal transcribed spacers of rDNA in diploid wheat Triticum urartu thum. ex. gandil].

Nucleotide sequences of 5.8S rRNA gene and rDNA internal transcribed spacers ITS-1 and ITS-2 were determined in diploid wheat Triticum urartu. It was shown that 5.8S rRNA gene of this wheat species consists of 163 base pairs and GC-content is 59.5%. When comparing 5.8S rRNA sequences in diploid wheat, rice and lupine and also 5.8S rRNA in hexaploid wheat and horse beans a high evolutional conservatism of its structure was revealed. The size of ITS-1 and ITS-2 in Tr. urartu is 219 and 225 base pairs long correspondingly. While comparing structures of similar rDNA regions of Tr. urartu, rice and maize a high level of homology was found only between nucleotides adjoining genes of high molecular rRNAs. In ITS-1 of Tr. urartu an insertion of 5'-GACGACGACATTGTCCGTC-3' was found, which is absent in maize and rice.

Base Sequence↗

PubMed2004

In vitro selection of RNA aptamers that bind to colicin E3 and structurally resemble the decoding site of 16S ribosomal RNA.

Colicin E3 is a ribonuclease that specifically cleaves at the site after A1493 of 16S rRNA in Escherichia coli ribosomes, thus inactivating translation. To analyze the interaction between colicin E3 and 16S rRNA, we used in vitro selection to isolate RNA ligands (aptamers) that bind to the C-terminal ribonuclease domain of colicin E3, from a degenerate RNA pool. Although the aptamers were not digested by colicin E3, they specifically bound to the protein (K(d) = 2-14 nM) and prevented the 16S rRNA cleavage by the C-terminal ribonuclease domain. Among these aptamers, aptamer F2-1 has a sequence similar to that of the region around the cleavage site from residue 1484 to 1506, including the decoding site, of E. coli 16S rRNA. The secondary structure of aptamer F2-1 was determined by the base pair covariation among the variants obtained by a second in vitro selection, using a doped RNA pool based on the aptamer F2-1 sequence. The sequence and structural similarities between the aptamers and 16S rRNA provide insights into the recognition of colicin E3 by this specific 16S rRNA region.

Base Sequence↗

PubMed1983

Primary sequence of the 5'-terminal region of mouse 18 S rRNA and adjacent spacer. Implications for rRNA processing.

Among the stepwise cleavage reactions involved in the processing of rRNA precursors in mammalian cells, an early event corresponds to the removal of the so-called 'external transcribed regions' which are located upstream 18 S rRNA sequence within the primary transcript. We have determined the primary sequence of the domain of mouse pre-rRNA which encompasses this early processing site and analyzed its structural features with reference to the other eukaryotic homologs available. The potential involvement of secondary structure features of rRNA precursors in the recognition process for cleavage is discussed.

Animals↗

PubMed2000

Using a targeted chemical nuclease to elucidate conformational changes in the E. coli 30S ribosomal subunit.

Determining the detailed tertiary structure of 16S rRNA within 30S ribosomal subunits remains a challenging problem. The particular structure of the RNA which allows tRNA to effectively interact with the associated mRNA during protein synthesis remains particularly ambiguous. This study utilizes a chemical nuclease, 1, 10-o-phenanthroline-copper, to localize regions of 16S rRNA proximal to the decoding region under conditions in which tRNA does not readily associate with the 30S subunit (inactive conformation), and under conditions which optimize tRNA binding (active conformation). By covalently attaching 1,10-phenanthroline-copper to a DNA oligomer complementary to nucleotides in the decoding region (1396-1403), we have determined that nucleotides 923-929, 1391-1396, and 1190-1192 are within approximately 15 A of the nucleotide base-paired to nucleotide 1403 in inactive subunits, but in active subunits only cleavages (1404-1405) immediately proximal to the 5' end of the hybridized probe remain. These results provide evidence for dynamic movement in the 30S ribosomal subunit, reported for the first time using a targeted chemical nuclease.

Base Sequence↗

PubMed2002

Crystallization and structure analysis of Thermus flavus 5S rRNA helix B.

The crystallization conditions of the synthetic RNA duplex r(GCGGCGU)*r(GCGCCGC), part of the Thermus flavus 5S rRNA domain B, were investigated in detail. The crystallization analysis revealed a relative narrow crystallization zone. Single sequence variations did not enhance the crystal quality, however the crystallization under microgravity provided crystals of higher quality. They belong to the space group P3(1)21 with unit cell dimensions of a = b = 35.0 A and c = 141.2 A. Diffraction data up to 2.6 A were collected and the structure subsequently analysed and refined to an R-value of 22.4 %. The conformation of the two molecules in the asymmetric unit is stabilized by intermolecular hydrogen bonds. The two molecules A and B are perpendicular to each other and interacting head to tail with symmetry related molecules. They form pseudo-continuous infinite helices in the crystal lattice.

Base Sequence↗

PubMed1999

Core sequence in the RNA motif recognized by the ErmE methyltransferase revealed by relaxing the fidelity of the enzyme for its target.

Under physiological conditions, the ErmE methyltransferase specifically modifies a single adenosine within ribosomal RNA (rRNA), and thereby confers resistance to multiple antibiotics. The adenosine (A2058 in Escherichia coli 23S rRNA) lies within a highly conserved structure, and is methylated efficiently, and with equally high fidelity, in rRNAs from phylogenetically diverse bacteria. However, the fidelity of ErmE is reduced when magnesium is removed, and over twenty new sites of ErmE methylation appear in E. coli 16S and 23S rRNAs. These sites show widely different degrees of reactivity to ErmE. The canonical A2058 site is largely unaffected by magnesium depletion and remains the most reactive site in the rRNA. This suggests that methylation at the new sites results from changes in the RNA substrate rather than the methyltransferase. Chemical probing confirms that the rRNA structure opens upon magnesium depletion, exposing potential new interaction sites to the enzyme. The new ErmE sites show homology with the canonical A2058 site, and have the consensus sequence aNNNcgGAHAg (ErmE methylation occurs exclusively at adenosines (underlined); these are preceded by a guanosine, equivalent to G2057; there is a high preference for the adenosine equivalent to A2060; H is any nucleotide except G; N is any nucleotide; and there are slight preferences for the nucleotides shown in lower case). This consensus is believed to represent the core of the motif that Erm methyltransferases recognize at their canonical A2058 site. The data also reveal constraints on the higher order structure of the motif that affect methyltransferase recognition.

Adenine↗