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At least 649 records · Page 36Linked to original sources

PubMed1988

Evidence for movement of the alpha-amylase gene into two phylogenetically distant Bacillus stearothermophilus strains.

The gene for an alpha-amylase cloned from strain DY-5 of Bacillus stearothermophilus was used to examine to what extent the corresponding genes are structurally similar in other B. stearothermophilus strains. The structure of the gene itself was almost identical in DY-5 and a group of strains represented by strain 799. The gene was not detected at all in strain DSM2334, which was phenotypically amylase deficient. Comparison of the structure of 5S rRNA and electrophoretic pattern of the ribosomal proteins indicates that strains DY-5 and DSM2334 are closely related to each other, whereas strain 799 is phylogenetically very distant from the two. We estimate that strain 799 separated from DY-5 and DSM2334 some 420 million years ago. Nucleotide sequencing of the region containing the amylase gene from strains DY-5 and 799 revealed the presence of a 3.4-kilobase stretch that was highly similar in the two strains. Furthermore, comparison of the restriction map surrounding the amylase gene of DY-5 with that of a corresponding region in DSM2334 indicated that the former strain contained an extra segment 5.5 kilobases in length, which included the 3.4-kilobase stretch mentioned above. This segment was missing in DSM2334. It thus appears that the alpha-amylase gene was brought into strains DY-5 and 799 from outside despite a large phylogenetic distance.

Base Sequence↗

PubMed1999

Structure and functions of nucleolin.

Nucleolin is an abundant protein of the nucleolus. Nucleolar proteins structurally related to nucleolin are found in organisms ranging from yeast to plants and mammals. The association of several structural domains in nucleolin allows the interaction of nucleolin with different proteins and RNA sequences. Nucleolin has been implicated in chromatin structure, rDNA transcription, rRNA maturation, ribosome assembly and nucleo-cytoplasmic transport. Studies of nucleolin over the last 25 years have revealed a fascinating role for nucleolin in ribosome biogenesis. The involvement of nucleolin at multiple steps of this biosynthetic pathway suggests that it could play a key role in this highly integrated process.

Animals↗

PubMed2006

Toward a census of bacteria in soil.

For more than a century, microbiologists have sought to determine the species richness of bacteria in soil, but the extreme complexity and unknown structure of soil microbial communities have obscured the answer. We developed a statistical model that makes the problem of estimating richness statistically accessible by evaluating the characteristics of samples drawn from simulated communities with parametric community distributions. We identified simulated communities with rank-abundance distributions that followed a truncated lognormal distribution whose samples resembled the structure of 16S rRNA gene sequence collections made using Alaskan and Minnesotan soils. The simulated communities constructed based on the distribution of 16S rRNA gene sequences sampled from the Alaskan and Minnesotan soils had a richness of 5,000 and 2,000 operational taxonomic units (OTUs), respectively, where an OTU represents a collection of sequences not more than 3% distant from each other. To sample each of these OTUs in the Alaskan 16S rRNA gene library at least twice, 480,000 sequences would be required; however, to estimate the richness of the simulated communities using nonparametric richness estimators would require only 18,000 sequences. Quantifying the richness of complex environments such as soil is an important step in building an ecological framework. We have shown that generating sufficient sequence data to do so requires less sequencing effort than completely sequencing a bacterial genome.

Alaska↗

PubMed2005

New insights into the interaction of ribosomal protein L1 with RNA.

The RNA-binding ability of ribosomal protein L1 is of profound interest, since L1 has a dual function as a ribosomal structural protein that binds rRNA and as a translational repressor that binds its own mRNA. Here, we report the crystal structure at 2.6 A resolution of ribosomal protein L1 from the bacterium Thermus thermophilus in complex with a 38 nt fragment of L1 mRNA from Methanoccocus vannielii. The conformation of RNA-bound T.thermophilus L1 differs dramatically from that of the isolated protein. Analysis of four copies of the L1-mRNA complex in the crystal has shown that domain II of the protein does not contribute to mRNA-specific binding. A detailed comparison of the protein-RNA interactions in the L1-mRNA and L1-rRNA complexes identified amino acid residues of L1 crucial for recognition of its specific targets on the both RNAs. Incorporation of the structure of bacterial L1 into a model of the Escherichia coli ribosome revealed two additional contact regions for L1 on the 23S rRNA that were not identified in previous ribosome models.

Amino Acid Sequence↗

PubMed1997

Structure of 18 S ribosomal RNA in native 40 S ribosomal subunits.

We have analyzed the structure of 18 S rRNA in native 40 S subunits using chemical modification followed by primer extension. The native subunits were modified using the single-stranded specific reagents dimethyl sulfate and 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide metho-p-toluenesulfonate. The modification pattern of the 18 S rRNA was compared to that obtained from derived 40 S subunits prepared by dissociation of unprogrammed 80 S ribosomes. Eighteen nucleotides showed different accessibility to the chemical probes in derived and native subunits. Half of these nucleotides were found in the central domain of the rRNA between the 1060 loop and the central pseudoknot. The remaining nucleotides were located in two clusters in the 5'- and 3'-domains of the 18 S rRNA. Derived 40 S subunits are free from non-ribosomal proteins. In contrast, native subunits are intermediates in protein synthesis initiation and contain stoichiometric amounts of initiation factor 3 (Sundkvist, I. C., and Staehelin, T. (1975) J. Mol. Biol. 99, 401-418). The possible role of this factor in altering the structure of 18 S rRNA in the native 40 S subunits is discussed.

Autoradiography↗

PubMed2001

Construction and analysis of base-paired regions of the 16S rRNA in the 30S ribosomal subunit determined by constraint satisfaction molecular modelling.

Structure models for each of the secondary structure regions from the Escherichia coli 16S rRNA (58 separate elements) were constructed using a constraint satisfaction modelling program to determine which helices deviated from classic A-form geometry. Constraints for each rRNA element included the comparative secondary structure, H-bonding conformations predicted from patterns of base-pair covariation, tertiary interactions predicted from covariation analysis, chemical probing data, rRNA-rRNA crosslinking information, and coordinates from solved structures. Models for each element were built using the MC-SYM modelling algorithm and subsequently were subjected to energy minimization to correct unfavorable geometry. Approximately two-thirds of the structures that result from the input data are very similar to A-form geometry. In the remaining instances, the presence of internal loops and bulges, some sequences (and sequence covariants) and accessory information require deviation from A-form geometry. The structures of regions containing more complex base-pairing arrangements including the central pseudoknot, the 530 region, and the pseudoknot involving base-pairing between G570-U571/A865-C866 and G861-C862/G867-C868 were predicted by this approach. These molecular models provide insight into the connection between patterns of H-bonding, the presence of unpaired nucleotides, and the overall geometry of each element.

Base Pairing↗

PubMed1995

Transcription in the yeast rRNA gene locus: distribution of the active gene copies and chromatin structure of their flanking regulatory sequences.

In growing yeast cells, about half of the 150 tandemly repeated rRNA genes are transcriptionally active and devoid of nucleosomes. By using the intercalating drug psoralen as a tool to mark accessible sites along chromatin DNA in vivo, we found that the active rRNA gene copies are rather randomly distributed along the ribosomal rRNA gene locus. Moreover, results from the analysis of a single, tagged transcription unit in the tandem array are not consistent with the presence of a specific subset of active genes that is stably maintained throughout cell divisions. In the rRNA intergenic spacers of yeast cells, an enhancer is located at the 3' end of each transcription unit, 2 kb upstream of the next promoter. Analysis of the chromatin structure along the tandem array revealed a structural link between transcription units and adjacent, 3' flanking enhancer sequences: each transcriptionally active gene is flanked by a nonnucleosomal enhancer, whereas inactive, nucleosome-packed gene copies are followed by enhancers regularly packaged in nucleosomes. From the fact that nucleosome-free enhancers were also detected in an RNA polymerase I mutant strain, we interpret these open chromatin structures as being the result of specific protein-DNA interactions that can occur before the onset of transcription. In contrast, in this mutant strain, all of the rRNA coding sequences are packaged in nucleosomal arrays. This finding indicates that the establishment of the open chromatin conformation on the activated gene copies requires elongating RNA polymerase I molecules advancing through the template.

Cell Division↗

PubMed1998

Role of histone H1 as an architectural determinant of chromatin structure and as a specific repressor of transcription on Xenopus oocyte 5S rRNA genes.

We explore the role of histone H1 as a DNA sequence-dependent architectural determinant of chromatin structure and of transcriptional activity in chromatin. The Xenopus laevis oocyte- and somatic-type 5S rRNA genes are differentially transcribed in embryonic chromosomes in vivo depending on the incorporation of somatic histone H1 into chromatin. We establish that this effect can be reconstructed at the level of a single nucleosome. H1 selectively represses oocyte-type 5S rRNA genes by directing the stable positioning of a nucleosome such that transcription factors cannot bind to the gene. This effect does not occur on the somatic-type genes. Histone H1 binds to the 5' end of the nucleosome core on the somatic 5S rRNA gene, leaving key regulatory elements in the promoter accessible, while histone H1 binds to the 3' end of the nucleosome core on the oocyte 5S rRNA genes, specifically blocking access to a key promoter element (the C box). TFIIIA can bind to the somatic 5S rRNA gene assembled into a nucleosome in the presence of H1. Because H1 binds with equivalent affinities to nucleosomes containing either gene, we establish that it is the sequence-selective assembly of a specific repressive chromatin structure on the oocyte 5S rRNA genes that accounts for differential transcriptional repression. Thus, general components of chromatin can determine the assembly of specific regulatory nucleoprotein complexes.

Animals↗

PubMed1996

Characterization of Bacterial Communities from Activated Sludge: Culture-Dependent Numerical Identification Versus In Situ Identification Using Group- and Genus-Specific rRNA-Targeted Oligonucleotide Probes

The structures of bacterial communities were studied in activated sludge samples obtained from the aerobic and anaerobic zones of a wastewater treatment plant showing enhanced phosphorous removal. Samples were analyzed by in situ hybridization with oligonucleotide probes complementary to selected regions of the 16S and 23S ribosomal RNA (rRNA) characteristic for defined phylogenetic entities (genera and larger groups). The microbial community structures revealed by molecular techniques were compared with the compositions of culturable bacterial communities, obtained from the characterization of 255 isolates from tryptone-soy (TS) agar and R2A agar. These isolates were characterized by 89 physiological tests and their cellular fatty acid patterns, and identified. Culture-dependent techniques indicated the following distribution: different Aeromonas spp. (2.7-8.3% on R2A agar; 45.0-63.7% on TS agar), Acinetobacter spp. (5.4-9.0% on R2A agar; 5.0-9.1% on TS agar), Pseudomonas spp. (up to 10% on R2A agar) and Shewanella putrefaciens (up to 3.0% on R2A agar), all members of the gamma subclass of Proteobacteria, were isolated most frequently. The relatively rare isolates of the beta subclass were identified as Acidovorax spp., Alcaligenes spp., and Comamonas spp.. The Gram-positive bacteria (high DNA G+C) were assigned mainly to Arthrobacter spp., Microbacterium spp., and Mycobacterium phlei. In order to assess the in situ abundance of the most frequently isolated genus, Aeromonas, two rRNA-targeted oligonucleotide probes were developed. The two gamma proteobacterial genera Aeromonas and Acinetobacter constituted less than 5% of all bacteria. In situ, Proteobacteria belonging to the beta subclass and high G+C Gram-positive bacteria were dominant. From filamentous bacteria, Sphaerotilus spp. and Leptothrix spp. could be detected occasionally. In addition, one sample contained a high proportion of the morphologically distinct filaments of Microthrix parvicella.

Journal Article↗

PubMed2005

Ribosomal protein L1 recognizes the same specific structural motif in its target sites on the autoregulatory mRNA and 23S rRNA.

The RNA-binding ability of ribosomal protein L1 is of profound interest since the protein has a dual function as a ribosomal protein binding rRNA and as a translational repressor binding its mRNA. Here, we report the crystal structure of ribosomal protein L1 in complex with a specific fragment of its mRNA and compare it with the structure of L1 in complex with a specific fragment of 23S rRNA determined earlier. In both complexes, a strongly conserved RNA structural motif is involved in L1 binding through a conserved network of RNA-protein H-bonds inaccessible to the solvent. These interactions should be responsible for specific recognition between the protein and RNA. A large number of additional non-conserved RNA-protein H-bonds stabilizes both complexes. The added contribution of these non-conserved H-bonds makes the ribosomal complex much more stable than the regulatory one.

Binding Sites↗

PubMed1999

ErmE methyltransferase recognizes features of the primary and secondary structure in a motif within domain V of 23 S rRNA.

The Erm methyltransferases confer resistance to macrolide, lincosamide and streptogramin B (MLS) antibiotics by methylation of a single adenosine base within bacterial 23 S ribosomal RNA. The ErmE methyltransferase, from the macrolide-producing bacterium Saccharopolyspora erythraea, recognizes a motif within domain V of the rRNA that specifically targets adenosine 2058 (A2058) for methylation. Here, we define the structure of the RNA motif by a combination of molecular genetics and biochemical probing. The core of the motif has the primary sequence 2056-GGAHA-2060, where H is any nucleotide except guanosine, and ErmE methylates at the adenosine in bold. For efficient recognition by ErmE, this sequence must be displayed within a particular secondary structure. An irregular stem (helix 73) is required immediately 5' to A2058, with an unpaired nucleotide, preferably a cytidine residue, at position 2055. Nucleotides 2611 to 2616 are collectively required to form part of the 3'-side of helix 73, but there is little or no restriction on the identities of individual nucleotides here. There are minor preferences in the identities of nucleotides 2051 to 2055 that are adjacent to the motif core, although their main role is in maintaining the irregular secondary structure. The essential elements of the ErmE motif are conserved in bacterial 23 S rRNAs, and thus presumably also form the recognition motif for other Erm methyltransferases.

Adenosine↗

PubMed1995

Effects of sequence alignment on the phylogeny of Sarcocystis deduced from 18S rDNA sequences.

The family Sarcocystidae contains a wide variety of parasitic protozoa, some of which are important pathogens of livestock and humans. The taxonomic relationships between two of the genera in this family (Toxoplasma and Sarcocystis) have been debated for a number of years and remain controversial. Recent studies, from comparisons of 18S rDNA-sequence data, have suggested that Sarcocystis is paraphyletic, although a hypothesis supporting monophyly of Sarcocystis could not be rejected. The present study shows that the phylogenetically informative nucleotide positions within the 18S rDNA are primarily located in the regions that make up the helices in the secondary structure of the 18S rRNA. A phylogenetic analysis of 18S rDNA-sequence data aligned by secondary structure constraints, or a subset of the data corresponding to all nucleotides found in the helices, provide unambiguous evidence supporting monophyly of Sarcocystis.

Animals↗

PubMed1991

Synthesis of large rRNAs by RNA polymerase II in mutants of Saccharomyces cerevisiae defective in RNA polymerase I.

The 35S rRNA gene of the yeast Saccharomyces cerevisiae was fused to the GAL7 promoter. This hybrid gene, when present on a multicopy plasmid and induced by galactose, suppressed the growth defects of a temperature-sensitive RNA polymerase I (pol I) mutant and those of a mutant in which the gene for the second largest subunit of pol I was deleted. Analysis of pulse-labeled RNA directly demonstrated that rRNA synthesis in this deletion mutant is from the GAL7 promoter. These experiments show that the sole essential function of pol I is the transcription of the rRNA genes, that pol I is not absolutely required for the synthesis of rRNA and ribosomes or cell growth if 35S rRNA synthesis is achieved by some other means, and that the tandemly repeated structure of the chromosomal rRNA genes is also not absolutely required for the synthesis of rRNA and ribosomes.

Base Sequence↗

PubMed1999

Ribosomal protein S14 of Saccharomyces cerevisiae regulates its expression by binding to RPS14B pre-mRNA and to 18S rRNA.

Production of ribosomal protein S14 in Saccharomyces cerevisiae is coordinated with the rate of ribosome assembly by a feedback mechanism that represses expression of RPS14B. Three-hybrid assays in vivo and filter binding assays in vitro demonstrate that rpS14 directly binds to an RNA stem-loop structure in RPS14B pre-mRNA that is necessary for RPS14B regulation. Moreover, rpS14 binds to a conserved helix in 18S rRNA with approximately five- to sixfold-greater affinity. These results support the model that RPS14B regulation is mediated by direct binding of rpS14 either to its pre-mRNA or to rRNA. Investigation of these interactions with the three-hybrid system reveals two regions of rpS14 that are involved in RNA recognition. D52G and E55G mutations in rpS14 alter the specificity of rpS14 for RNA, as indicated by increased affinity for RPS14B RNA but reduced affinity for the rRNA target. Deletion of the C terminus of rpS14, where multiple antibiotic resistance mutations map, prevents binding of rpS14 to RNA and production of functional 40S subunits. The emetine-resistant protein, rpS14-EmRR, which contains two mutations near the C terminus of rpS14, does not bind either RNA target in the three-hybrid or in vitro assays. This is the first direct demonstration that an antibiotic resistance mutation alters binding of an r protein to rRNA and is consistent with the hypothesis that antibiotic resistance mutations can result from local alterations in rRNA structure.

Alkaloids↗

PubMed1994

A common core structure for U3 small nucleolar RNAs.

U3 nucleolar small RNA (snRNA) is involved in early processing of the primary rRNA transcript. A secondary structure model for the unusually small Trypanosoma brucei U3 snRNA was deduced by chemical modification and enzymatic cleavage of U3 snRNA in deproteinized and ribonucleoprotein (RNP) forms. Comprehensive alignment of U3 snRNAs from vertebrate, plant, fungal and protozoan species clearly delineated conserved and divergent features. The 5' domain of the T. brucei U3 snRNA appears to form one small, flexible 5' stem loop structure followed by a long single-stranded region; this model is a variation on 5' domain structures proposed for other U3 snRNAs which do not conform to a single model. The 3' domain of T. brucei U3 snRNA contains four single-stranded sequences conserved between U3 snRNAs. Of these, structural probing determined that the configurations of GAU region and box B and C sequences are altered by protein interactions in U3 snRNP. Conspicuously, the 3' domains of trypanosomal U3 snRNAs lack stem loops II and III, indicating that these structures are not required for conserved U3 snRNA functions.

Animals↗

PubMed1990

Structural analysis of the 5' domain of the HeLa 18S ribosomal RNA by chemical and enzymatic probing.

The secondary structure of HeLa 18S rRNA was investigated by a combination of chemical and enzymatic probing techniques. Using four chemical reagents (DMS*, kethoxal, DEPC and CMCT) which react specifically with unpaired bases and two nucleases (RNase T1 and cobra venom nuclease) which cleave the ribopolynucleotides at unpaired guanines and helical segments, we have analyzed the secondary structure of the 5' domain of 18S rRNA isolated from HeLa 40S ribosomal subunits. The sites at which chemical modifications and nuclease cleavages occurred were identified by primer extension using synthetic deoxyoligonucleotides and reverse transcriptase. These studies led to the deduction of an intra-RNA pairing pattern from the available secondary structure models based on comparative sequence analysis. Apart from the general canonical pairing we have identified noncanonical U-U, G-A, A-G, A-C, C-A and G-G pairing in HeLa 18S rRNA. The differential reactivity of bases to chemical reagents has enabled us to predict the possible configuration of these bases in some of the noncanonical pairing. The absence of chemical reactivities and cobra venom nuclease sensitivity in the terminal loops of helices 6 and 12 indicate a tertiary interaction unique to HeLa 18S rRNA. We have confirmed the existence of the complex tertiary folding recently proposed (Gutell and Woese 1990 Proc. Natl. Acad. Sci. 87, 663-667) for the universally conserved helix 19 in HeLa 18S rRNA. The complementarity of chemical modifications and enzymatic cleavages provided experimental evidence for the proposal of a model structure for the 655 nucleotides of the 5' domain of HeLa 18S rRNA.

Base Sequence↗

PubMed1995

DNA sequence, structure, and phylogenetic relationship of the mitochondrial small-subunit rRNA from the red alga Chondrus crispus (Gigartinales rhodophytes).

The entire nucleotide sequence containing the small-subunit ribosomal RNA gene (SSU rRNA) from the mitochondrial genome of Chondrus crispus was determined. To our knowledge, this is the first sequence of a mitochondrial 16S-like rRNA from a red alga. The length of this gene is 1,376 nucleotides. Its secondary structure was constructed and compared with other known secondary structures from eubacteria and from mitochondria of land plants, green and brown algae, and fungi. Phylogenetic trees were built upon SSU rRNA sequence alignment from mitochondria and eubacteria. The results show that rhodophytes and chromophytes provide additional links in the evolution of mitochondria between the green plant lineage and the "nonplant" lineages.

Base Sequence↗

PubMed2001

Molecular organization of the ribosomal RNA transcription unit and the phylogenetic study of Zymomonas mobilis ZM4.

Previously we reported that Zymomonas mobilis ZM4 contains three ribosomal transcription units (rrnA to C operons) which are clustered around the 50 min region, PacI fragments 13 and 6, on the physical map of Z. mobilis ZM4 [Kang, H. L. and Kang, H. S. (1998) Gene 206, 223-228]. The physical map reveals that the rrnA gene set is located on the 76 kb PacI fragment 13. The complete nucleotide sequence of the rrnA gene set has been determined. The total number of nucleotides of the rrnA gene set is about 6,250 bp. The structural genes of the 16S, 23S and 5S rRNA code for the 1,478 nt, 2,786 nt and 121 nt RNA chains, respectively. The length of the spacer regions between the 16S and 23S rRNA genes, and between the 23S and 5S rRNA genes, are 606 bp and 101 bp, respectively. Two tRNA genes, Ile-tRNA and Ala-tRNA, are found between the 16S and 23S rRNA genes and a fMet-tRNA gene is identified downstream of the 5S rRNA gene. Thus, the molecular organization of this rrnA gene set is the order of 5'-16S rRNA-tRNAIle-tRNAAla-23S rRNA-5S rRNA-tRNAfMet-3'. The secondary structure models of 16S, 23S and 5S rRNA are proposed. The phylogenetic tree, based on the 16S rRNA sequence, was constructed by neighbor-joining and maximum-parsimony methods. Zymomonas belongs to a group in which Aqorbacterium, Rhodobacter and Sphingomonas are included.

Base Sequence↗