PubMed Health⌕ Search

SEARCH · PubMed Health

Results for “RNA, BACTERIAL”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 73 records · Page 4Linked to original sources
PubMed2004

Establishment of a real-time PCR-based approach for accurate quantification of bacterial RNA targets in water, using Salmonella as a model organism.

Quantitative PCR (Q-PCR) is a fast and efficient tool to quantify target genes. In eukaryotic cells, quantitative reverse transcription-PCR (Q-RT-PCR) is also used to quantify gene expression, with stably expressed housekeeping genes as standards. In bacteria, such stable expression of housekeeping genes does not occur, and the use of DNA standards leads to a broad underestimation. Therefore, an accurate quantification of RNA is feasible only by using appropriate RNA standards. We established and validated a Q-PCR method which enables the quantification of not only the number of copies of target genes (i.e., the number of bacterial cells) but also the number of RNA copies. The genes coding for InvA and the 16S rRNA of Salmonella enterica serovar Typhimurium were selected for the evaluation of the method. As DNA standards, amplified fragments of the target genes were used, whereas the same DNA standards were transcribed in vitro for the development of appropriate RNA standards. Salmonella cultures and environmental water samples inoculated with bacteria were then employed for the final testing. Both experimental approaches led to a sensitive, accurate, and reproducible quantification of the selected target genes and RNA molecules by Q-PCR and Q-RT-PCR. It is the first time that RNA standards have been successfully used for a precise quantification of the number of RNA molecules in prokaryotes. This demonstrates the potential of this approach for determining the presence and metabolic activity of pathogenic bacteria in environmental samples.

Bacterial Proteins↗
PubMed1994

Human RNA polymerase II subunit hRPB14 is homologous to yeast RNA polymerase I, II, and III subunits (AC19 and RPB11) and is similar to a portion of the bacterial RNA polymerase alpha subunit.

The cDNA cloning of the human polII 14-kDa subunit, hRPB14, and the comparison of its aa sequence with those of other pol subunits are described. The aa sequence of hRPB14 has homology to yeast poIII subunit RPB11 (44%), to a common subunit of yeast polI and polIII AC19 (24%) and to a Caenorhabditis elegans sequence (33%). hRPB14 contains a 19-aa motif, located in its N terminus, which was also found in human polII 33-kDa subunit hRPB33, yeast pol subunits (AC40, AC19, RPB3 and RPB11), and in the bacterial pol alpha subunit, which was involved in subunit assembly. This motif was also conserved in the conjugation-specific gene products of Tetrahymena (CnjC), Merchantia polymorpha chloroplast DNA (RNLVA) and C. elegans DNA (CEF58A4; deduced from the nucleotide sequence and of unknown function). The evolutionary emergence of a probable eukaryotic heterodimer, hRPB14/hRPB33, from a prokaryotic homodimer, alpha 2, is hypothesized.

Amino Acid Sequence↗
PubMed1981

Antigenic variability of bacterial RNA polymerases.

Radioimmunoassay analysis of enteric and some other Gram-negative bacteria has shown that the antigenic structure of the RNA polymerase alpha subunit is more conserved than that of the beta and beta' subunits. Since anti-alpha antibodies do not affect RNA polymerase activity, the constraints which determine the low variability of the antigenic structure of the alpha subunit are not directly related to its functional role. The antigenic determinants of the alpha subunit located on the surface of the RNA polymerase molecule are more conserved than those involved in contacts with other subunits; an opposite tendency characterizes the beta subunit. The range of variability of the antigenic determinants buried inside the RNA polymerase molecule suggests that the subunits are attached to each other rather loosely. Immunological comparison of RNA polymerases provides a simple method for reconstructing bacterial genealogies. The genealogy of the bacteria examined is essentially in agreement with phylogenetic trees based on 16S and 5S rRNA sequence characterization. This argues against extensive interspecific transfer of genes coding for components of the transcription and translation apparatus.

Bacillaceae↗
PubMed2002

Structure of the bacterial RNA polymerase promoter specificity sigma subunit.

The sigma subunit is the key regulator of bacterial transcription. Proteolysis of Thermus aquaticus sigma(A), which occurred in situ during crystallization, reveals three domains, sigma(2), sigma(3), and sigma(4), connected by flexible linkers. Crystal structures of each domain were determined, as well as of sigma(4) complexed with -35 element DNA. Exposed surfaces of each domain are important for RNA polymerase binding. Universally conserved residues important for -10 element recognition and melting lie on one face of sigma(2), while residues important for extended -10 recognition lie on sigma(3). Genetic studies correctly predicted that a helix-turn-helix motif in sigma(4) recognizes the -35 element but not the details of the protein-DNA interactions. Positive control mutants in sigma(4) cluster in two regions, positioned to interact with activators bound just upstream or downstream of the -35 element.

Bacterial Proteins↗
PubMed1981

Identification of transcription initiation sites for bacterial RNA polymerase and eukaryotic RNA polymerase B on the 5' end of the mouse beta-Globin gene.

Using a recombinant phage containing the mouse beta-Globin gene with lambda gtWES bacteriophage DNA, transcription initiation sites for Escherichia coli RNA polymerase and calf thymus RNA polymerase B were mapped at the 5' and 3' ends of the mouse beta-Globin gene. The bacterial enzyme was capable of initiating RNA synthesis at the 3' end site located at about 700 residues from the 3' end of the beta-Globin restriction enzyme map. Initiation at this site was more efficient than initiation at the known early lambda promotors (PL, PR). Calf thymus RNA polymerase B initiated transcription at the same sites as the bacterial enzyme but in this case maximum efficiency was at the 5' end site as compared to the 3' end site. Initiation of transcription occurs in the region of the d(T-A-T-A-A) sequence. Initiation efficiency at the 5' end site, as probed by the maximum rate of transcription, was shown to depend partly upon the presence of the adjacent sequences upstream and downstream of the 5' initiation site.

Base Sequence↗
PubMed2003

A new class of bacterial RNA polymerase inhibitor affects nucleotide addition.

RNA polymerase (RNAP) is the central enzyme of gene expression. Despite availability of crystal structures, details of its nucleotide addition cycle remain obscure. We describe bacterial RNAP inhibitors (the CBR703 series) whose properties illuminate this mechanism. These compounds inhibit known catalytic activities of RNAP (nucleotide addition, pyrophosphorolysis, and Gre-stimulated transcript cleavage) but not translocation of RNA or DNA when translocation is uncoupled from catalysis. CBR703-resistance substitutions occur on an outside surface of RNAP opposite its internal active site. We propose that CBR703 compounds inhibit nucleotide addition allosterically by hindering movements of active site structures that are linked to the CBR703 binding site through a bridge helix.

Amidines↗
PubMed1982

Genetic analysis of two bacterial RNA polymerase mutants that inhibit the growth of bacteriophage T7.

The Escherichia coli mutants 7009 and BR3 are defective in the growth of bacteriophage T7. We have previously shown that both of these mutant hosts produce an altered RNA polymerase which is resistant to inhibition by the T7 gene 2 protein (De Wyngaert and Hinkle 1979). In both strains, the mutation which prevents T7 growth is closely linked to rifA (rpoB). Both mutants are complemented by transformation with a multicopy plasmid carrying rpoB and rpoC but not by a plasmid carrying only rpoB. This indicates that the mutations reside in rpoC, the structural gene for the beta' subunit of RNA polymerase. When a single copy of the wildtype rpoC allele is introduced into the mutant using the transducing phage lambda drifd18, the mutant allele is dominant over wildtype. The lambda drifd18 transductant also remains unable to support the growth of T7 in the presence of rifampin. This supports our conclusion that the mutation is in rpoC. We have measured the growth of T7 phage, the kinetics of phage DNA synthesis, and the structure of replicative DNA intermediates in several transductants, and compared these results with those obtained in the original mutant strains.

DNA↗
PubMed2005

A phylogeny of bacterial RNA nucleotidyltransferases: Bacillus halodurans contains two tRNA nucleotidyltransferases.

We have analyzed the distribution of RNA nucleotidyltransferases from the family that includes poly(A) polymerases (PAP) and tRNA nucleotidyltransferases (TNT) in 43 bacterial species. Genes of several bacterial species encode only one member of the nucleotidyltransferase superfamily (NTSF), and if that protein functions as a TNT, those organisms may not contain a poly(A) polymerase I like that of Escherichia coli. The genomes of several of the species examined encode more than one member of the nucleotidyltransferase superfamily. The function of some of those proteins is known, but in most cases no biochemical activity has been assigned to the NTSF. The NTSF protein sequences were used to construct an unrooted phylogenetic tree. To learn more about the function of the NTSFs in species whose genomes encode more than one, we have examined Bacillus halodurans. We have demonstrated that B. halodurans adds poly(A) tails to the 3' ends of RNAs in vivo. We have shown that the genes for both of the NTSFs encoded by the B. halodurans genome are transcribed in vivo. We have cloned, overexpressed, and purified the two NTSFs and have shown that neither functions as poly(A) polymerase in vitro. Rather, the two proteins function as tRNA nucleotidyltransferases, and our data suggest that, like some of the deep branching bacterial species previously studied by others, B. halodurans possesses separate CC- and A-adding tRNA nucleotidyltransferases. These observations raise the interesting question of the identity of the enzyme responsible for RNA polyadenylation in Bacillus.

Amino Acid Sequence↗
PubMed2000

Pausing by bacterial RNA polymerase is mediated by mechanistically distinct classes of signals.

Transcript elongation by RNA polymerase is discontinuous and interrupted by pauses that play key regulatory roles. We show here that two different classes of pause signals punctuate elongation. Class I pauses, discovered in enteric bacteria, depend on interaction of a nascent RNA structure with RNA polymerase to displace the 3' OH away from the catalytic center. Class II pauses, which may predominate in eukaryotes, cause RNA polymerase to slide backwards along DNA and RNA and to occlude the active site with nascent RNA. These pauses differ in their responses to antisense oligonucleotides, pyrophosphate, GreA, and general elongation factors NusA and NusG. In contrast, substitutions in RNA polymerase that increase or decrease the rate of RNA synthesis affect both pause classes similarly. We propose that both pause classes, as well as arrest and termination, arise from a common intermediate that itself binds NTP substrate weakly.

DNA-Directed RNA Polymerases↗
PubMed1990

Identification of transcription promoter regions from rat mtDNA that are utilized in vivo by the bacterial RNA polymerase.

We have used a prokaryotic promoter-identification vector, pKO-1, to isolate rat mitochondrial DNA (mtDNA) sequences that can act as bacterial transcription promoters. Three putative promoter-containing clones that hydridized to mtDNA probes were identified. The strength of the promoters was quantitated by measuring galactokinase activity. The three promoters mapped to three distinct regions of the mtDNA - one within the 5' half of the 16S rRNA gene, one within the ATPase subunit 6 gene, and the last at the carboxy terminal end of the cytochrome oxidase subunit I gene.

Adenosine Triphosphatases↗
PubMed1989

The plastid rpoA gene encoding a protein homologous to the bacterial RNA polymerase alpha subunit is expressed in pea chloroplasts.

The gene rpoA, encoding a protein homologous to the alpha subunit of RNA polymerase from Escherichia coli has been located in pea chloroplast DNA downstream of the petD gene for subunit IV of the cytochrome b-f complex. Nucleotide sequence analysis has revealed that rpoA encodes a polypeptide of 334 amino acid residues with a molecular weight of 38916. Northern blot analysis has shown that rpoA is co-transcribed with the gene for ribosomal protein S11. A lacZ-rpoA gene-fusion has been constructed and expressed in E. coli. Antibodies raised against the fusion protein have been employed to demonstrate the synthesis of the rpoA gene product in isolated pea chloroplasts. Western blot analysis using these antibodies and antibodies against the RNA polymerase core enzyme from the cyanobacterium, Anabaena 7120, has revealed the presence of the gene product in a crude RNA polymerase preparation from pea chloroplasts.

Amino Acid Sequence↗
PubMed2002

Mutations of bacterial RNA polymerase leading to resistance to microcin j25.

A mutation in the conserved segment of the rpoC gene, which codes for the largest RNA polymerase (RNAP) subunit, beta', was found to make Escherichia coli cells resistant to microcin J25 (MccJ25), a bactericidal 21-amino acid peptide active against Gram-negative bacteria (Delgado, M. A., Rintoul, M. R., Farias, R. N., and Salomon, R. A. (2001) J. Bacteriol. 183, 4543-4550). Here, we report that mutant RNAP prepared from MccJ25-resistant cells, but not the wild-type RNAP, is resistant to MccJ25 in vitro, thus establishing that RNAP is a true cellular target of MccJ25. We also report the isolation of additional rpoC mutations that lead to MccJ25 resistance in vivo and in vitro. The new mutations affect beta' amino acids in evolutionarily conserved segments G, G', and F and are exposed into the RNAP secondary channel, a narrow opening that connects the enzyme surface with the catalytic center. We also report that previously known rpoB (RNAP beta subunit) mutations that lead to streptolydigin resistance cause resistance to MccJ25. We hypothesize that MccJ25 inhibits transcription by binding in RNAP secondary channel and blocking substrate access to the catalytic center.

Amino Acid Sequence↗