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At least 487 records · Page 27Linked to original sources
PubMed2000

Isomerization of a binary sigma-promoter DNA complex by transcription activators.

Multisubunit RNA polymerases are targets of sophisticated signal transduction pathways that link environmental or temporal cues to changes in gene expression. Here we show that the sigma 54 protein (sigma54), responsible for promoter specific binding by bacterial RNA polymerase, undergoes a nucleotide hydrolysis dependent isomerization on DNA. Changes in protein structure are evident. The isomerization has all the known requirements of sigma 54-dependent transcription, including a dependence on enhancer binding activator proteins and occurs independently of the core RNA polymerase. We suggest that activator driven changes in sigma54 conformation trigger the conversion of a transcriptionally silent RNA polymerase conformation to one able to interact productively with template DNA. Our results illustrate the types of changes that must occur for multisubunit complexes to manipulate DNA, and show that transcription activators can remodel key nucleoprotein structures to achieve direct activation of transcription.

Bacterial Proteins↗
PubMed1999

Probing the interaction of T7 RNA polymerase with promoter.

Transcription is the fundamental process by which RNA is synthesized by RNA polymerases on double-stranded DNA templates. One structurally simple RNA polymerase is encoded by bacteriophage T7. T7 RNA polymerase is an excellent candidate for studying structural aspects of transcription, because unlike the eucaryotic and bacterial RNA polymerases, it is a single subunit enzyme and does not require additional factors to carry out the entire process of transcription from start to finish. An important advantage of studying transcription using this enzyme is that the high-resolution crystal structure of T7 RNA polymerase has been solved. However, a cocrystal structure of the polymerase complexed with promoter has not yet been published. Here, we have used cross-linking techniques to understand the interaction of promoter with T7 RNA polymerase. We constructed promoters that were substituted with the photo-cross-linkable nucleotide 5-iodo uracil at every dT in the promoter from -17 to -1. This substitution replaces the 5-methyl in dT with an iodine atom. The substituted promoters were photo-cross-linked to T7 RNAP, and the efficiency of cross-linking was quantitated at every position. In the melting domain, the strongest contacts occurred at -3 and at -1 on the template strand while very weak cross-linking was seen at -2 and at -4 on the nontemplate strand. In the binding domain, the strongest contacts were seen at -16, -15, and -13 and at -10 on the template strand while at -17 and -14 on the nontemplate strand very weak cross-linking was observed. Cross-linking was poor in the intervening region between the binding and the melting domains. These results suggested that, in the T7 RNA polymerase-promoter complex, the polymerase molecule mainly contacts the template bases in the TATA box while the upstream contacts are used as an anchor for DNA binding. For a systematic study designed to probe the nature of base-specific interactions in the polymerase-promoter complex, we used neutral salts from the Hofmeister series. In general, the order of perturbation was sulfate > citrate > acetate for anions and ammonium > magnesium > potassium for cations. Using acrylamide, a neutral hydrophobic agent to probe for nonionic contacts, we observed that at -2, -4, and -17 the contacts had a hydrophobic component, while at many other positions there was no significant effect, suggesting that the contacts in the promoter-polymerase complexes were predominantly ionic but at certain positions nonionic interactions also existed. To localize a specific interaction in the melting domain, we proteolyzed the cross-linked T7 RNAP and analyzed the fragments using gel electrophoresis, mass spectrometry, and amino acid composition. High-resolution mapping indicated that amino acid residues 614-627 may be in the vicinity of the melting domain. Specifically, Y623 may contact -3 on the template strand.

Bacteriophage T7↗
PubMed1999

Motility of Helicobacter pylori is coordinately regulated by the transcriptional activator FlgR, an NtrC homolog.

sigma54 is the subunit of bacterial RNA polymerase that transcribes from promoters with enhancer elements bound by enhancer-binding proteins. By computer searches of Helicobacter pylori genomic sequences, chromosomal gene disruption, and RNA analyses, we have identified sigma54-recognized promoters that regulate transcription of flagellar basal body and hook genes, as well as the enhancer-binding protein FlgR (flagellum regulator), a transactivating protein of the NtrC family. We demonstrate that FlgR is required for bacterial motility and transcription of five promoters for seven basal body and hook genes. In addition, FlgR acts as a repressor of transcription of the sigma28-regulated flaA flagellin gene promoter, while changes in DNA topology repress transcription of the sigma54-regulated flaB flagellin gene promoter. Our data indicate that regulation of flagellar gene expression in H. pylori shows similarities with that in enterobacteriaceae and Caulobacter.

Bacterial Proteins↗
PubMed1980

Evidence for direct involvement of T7 RNA polymerase bacteriophage DNA replication.

Experiments in a number of different systems have suggested that the initiation of DNA replication is often dependent upon transcription at the origin of replication. During infection with bacteriophage T7, the T7 genome is transcribed first by the bacterial RNA polymerase and then by a phage-coded enzyme, the product of gene 1. The bacterial enzyme does not appear to be directly involved in the initiation of replication as phage DNA synthesis is not inhibited by rifampin. For testing whether the T7 RNA polymerase plays a role in replication, a T7 gene 1 temperature-sensitive mutant was used, and the RNA polymerase was inactivated at intervals after infection by rapidly raising the temperature of the culture. The experiments indicated that the inactivation of the T7 RNA polymerase caused the cessation of phage DNA synthesis, even at later times during infection when the inhibition of protein synthesis with chloramphenicol had no effect on DNA replication. This suggests that in addition to its role in gene expression, the T7 RNA polymerase plays a direct role in T7 DNA replication.

Chloramphenicol↗
PubMed1999

RNA polymerase II as a control panel for multiple coactivator complexes.

1999 marks the 30th anniversary of the reported discovery of sigma factor and the bacterial RNA polymerase holoenzyme. In 1994, an RNA polymerase II complex was discovered in yeast - mammalian complexes were subsequently identified. Recent developments regarding the composition and function of RNA polymerase II complexes suggest, however, that the concept of the holoenzyme, as defined in bacteria, might not be relevant to eukaryotes.

Animals↗
PubMed1987

DNA sequence analysis suggests that expression of flagellar and chemotaxis genes in Escherichia coli and Salmonella typhimurium is controlled by an alternative sigma factor.

Biosynthesis of bacterial flagella involves the coordinated expression of 30 or more genes in several separate operons. We have recently shown that in Bacillus subtilis, the sigma 28 factor is essential for flagellar synthesis, suggesting that transcription of these genes is directly under the control of this alternative sigma factor. In enteric bacteria structural genes for flagellar, chemotaxis, and motility operons appear to be under coordinate control, however, the nature of the regulatory factors has not been determined. Sequence analysis of many such genes has failed to reveal plausible promoter sequences for the predominant bacterial RNA polymerase, and several such genes are not transcribed effectively in vitro by the Escherichia coli sigma 70 RNA polymerase. However, all of the sequenced flagellar, chemotaxis, and motility operons from the enteric bacteria are preceded by DNA sequences highly homologous to B. subtilis sigma 28 promoters. We propose that an alternative sigma factor controls expression of the flagellar regulon in both B. subtilis and in the enteric bacteria.

Bacterial Proteins↗
PubMed1999

Reverse transcription polymerase chain reaction method for the detection of glycopeptide resistance in enterococci.

In this work we have developed reverse transcription polymerase chain reaction (RT-PCR) methods for detecting specific mRNA from enterococci, particularly vanA and vanB genes, responsible for glycopeptide resistance in this genus. mRNA from the two genes was detected immediately after RNA extraction of a midlog phase culture, determined by growth rate analysis. Because of the short half-life associated with many bacterial RNA species (1.5-2 min), time is an important factor in obtaining RNA of good yield and high purity. Our results showed that: (i) the transcription of mRNA related to vanA ligase in enterococci showing Van A phenotype happens only after induction with both vancomycin and teicoplanin; (ii) the transcription of mRNA related to vanB ligase happens only in the presence of vancomycin and (iii) there was no transcription of mRNA in the two strains positive to vanA gene after PCR experiments. RT-PCR methodology can have numerous applications in microbiology for studying gene expression in isolated bacteria and also in nonculturable cells in environmental samples, for studies of mechanisms and/or as an indicator of viability in bacterial communities.

Anti-Bacterial Agents↗
PubMed1989

Sequence-dependent termination of bacteriophage T7 transcription in vitro by DNA-binding drugs.

An in vitro T7 bacteriophage transcription system has been utilized in which the RNA was initiated to a specific length (defined by the absence of the appropriate nucleoside triphosphate). When the DNA-RNA-RNA polymerase ternary complex was exposed to nonsaturating levels of DNA-binding ligands (i.e., a small fractional occupancy at each site), and the RNA transcript then allowed to elongate in the presence of all four nucleoside triphosphates, there was a synchronous increase of RNA lengths up to sites occupied by ligands. A unique characteristic is that bacteriophage transcription was completely terminated at every ligand site, in contrast to bacterial RNA polymerases where "read-through" past drug sites occurs and results merely in a delay of transcription at each site due primarily to dissociation of drug from the DNA. Similar termination of transcription at each drug site was observed with T3 and SP6 RNA polymerases. The termination at drug sites in the bacteriophage system results in RNA of specific lengths which define the location of ligand sites, and the RNA concentration provides a measure of relative ligand occupancy at that site. Termination of transcription was observed with four drugs with relatively long DNA residence times (half-life greater than or equal to 300 s at 20 degrees C for nogalamycin, actinomycin, mithramycin, and echinomycin) but to a lesser extent with drugs of intermediate residence times [a bis(thiadaunomycin) and an acridine-tripyrrole, with half-lives of 230 and 7 s, respectively, at 20 degrees C]

Anti-Bacterial Agents↗
PubMed1997

RNA polymerase sigma factor determines start-site selection but is not required for upstream promoter element activation on heteroduplex (bubble) templates.

Sequence-selective transcription by bacterial RNA polymerase (RNAP) requires sigma factor that participates in both promoter recognition and DNA melting. RNAP lacking sigma (core enzyme) will initiate RNA synthesis from duplex ends, nicks, gaps, and single-stranded regions. We have used DNA templates containing short regions of heteroduplex (bubbles) to compare initiation in the presence and absence of various sigma factors. Using bubble templates containing the sigmaD-dependent flagellin promoter, with or without its associated upstream promoter (UP) element, we demonstrate that UP element stimulation occurs efficiently even in the absence of sigma. This supports a model in which the UP element acts primarily through the alpha subunit of core enzyme to increase the initial association of RNAP with the promoter. Core and holoenzyme do differ substantially in the template positions chosen for initiation: sigmaD restricts initiation to sites 8-9 nucleotides downstream of the conserved -10 element. Remarkably, sigmaA also has a dramatic effect on start-site selection even though the sigmaA holoenzyme is inactive on the corresponding homoduplexes. The start sites chosen by the sigmaA holoenzyme are located 8 nucleotides downstream of sequences on the nontemplate strand that resemble the conserved -10 hexamer recognized by sigmaA. Thus, sigmaA appears to recognize the -10 region even in a single-stranded state. We propose that in addition to its described roles in promoter recognition and start-site melting, sigma also localizes the transcription start site.

Bacillus subtilis↗
PubMed1988

Genes coding for RNA polymerase beta subunit in bacteria. Structure/function analysis.

The nucleotide sequence of the rpoB gene of Salmonella typhimurium has been determined in this work. It was compared with known sequences of the gene from other sources and the conservative regions were detected. This allowed some interesting conclusions to be made about the distribution of the functional domains in bacterial RNA polymerase and about the three-dimensional structure of its beta subunit.

Amino Acid Sequence↗
PubMed2003

Exploitation of a chemical nuclease to investigate the location and orientation of the Escherichia coli RNA polymerase alpha subunit C-terminal domains at simple promoters that are activated by cyclic AMP receptor protein.

The C-terminal domain of the alpha subunit (alphaCTD) of bacterial RNA polymerase plays an important role in promoter recognition. It is known that alphaCTD binds to the DNA minor groove at different locations at different promoters via a surface-exposed determinant, the 265 determinant. Here we describe experiments that permit us to determine the location and orientation of binding of alphaCTD at any promoter. In these experiments, a DNA cleavage reagent is attached to specific locations on opposite faces of the RNA polymerase alpha subunit. After incorporation of the tagged alpha subunits into holo-RNA polymerase, patterns of DNA cleavage due to the reagent are determined in open complexes. The locations of DNA cleavage due to the reagent attached at different positions allow the position and orientation of alphaCTD to be deduced. Here we present data from experiments with simple Escherichia coli promoters that are activated by the cyclic AMP receptor protein.

Base Sequence↗
PubMed2003

A structural and functional study of plastid RNAs homologous to catalytic bacterial RNase P RNA.

Ribonuclease P (RNase P), the ubiquitous enzyme required for 5' maturation of transfer RNA, is a ribonucleoprotein containing an essential RNA subunit. This RNA (P RNA) is the catalytic component of RNase P in Bacteria and some Archaea. A putative P RNA is encoded in the chloroplast genome of three algae: Cyanophora paradoxa, Porphyra purpurea and Nephroselmis olivacea. In no case, the P RNAs from the plastids were active in vitro in conditions where bacterial and some archaeal P RNAs are functional. By using lead-ion-induced hydrolysis, we conclude that the catalytic deficiency is most likely due to the perturbation of the global structure of the plastid P RNAs compared to the bacterial counterpart. As a consequence, the plastid P RNAs are unable to bind to the precursor tRNA substrates. We discuss these results in the context of plastid and RNase P evolution.

Base Sequence↗
PubMed2000

Promoter opening by sigma(54) and sigma(70) RNA polymerases: sigma factor-directed alterations in the mechanism and tightness of control.

Transcription control at the melting step is not yet understood. Here, band shift, cross-linking, and transcription experiments on diverse DNA probes were used with two bacterial RNA polymerase holoenzymes that differ in how they regulate melting. Data indicated that both sigma(54) and sigma(70) holoenzymes assume a default closed form that cannot establish single-strand binding. Upon activation the enzymes are converted to an open form that can bind simultaneously to the upstream fork junction and to the melted transcription start site. The key difference is that sigma(54) imposes tighter regulation by creating a complex molecular switch at -12/-11; the current data show that this switch can be thrown by activator. In this case an ATP-bound enhancer protein causes sigma(54) to alter its cross-linking pattern near -11 and also causes a reorganization of holoenzyme: DNA interactions, detected by electrophoretic mobility-shift assay. At a temperature-dependent sigma(70) promoter, elevated temperature alone can assist in triggering conformational changes that enhance the engagement of single-strand DNA. Thus, the two sigma factors modify the same intrinsic opening pathway to create quite different mechanisms of transcriptional regulation.

Adenine↗
PubMed1982

[Effect of nalidixic acid on the activation of RNA synthesis in outgrowing spores of Bacillus subtilis].

Outgrowth of B. subtilis spores depends on the action of DNA gyrase (comp. Matsuda and Kameyama 1980). Application of nalidixic acid (100 micrograms/ml) to dormant spores of Bacillus subtilis prevents the outgrowth. Application of nalidixic acid (100 micrograms/ml) during the early outgrowth phase (after a 20 min germination period) does not prevent, but only delay spore outgrowth. Germination of spores is not influenced. Nalidixic acid is an effective inhibitor of RNA synthesis in outgrowing spores, whereas vegetative cells are more resistant. Spores can grow out inspite of a remarkably reduced intensity of RNA synthesis. Nalidixic acid particularly inhibits the synthesis of stable RNA, probably that of ribosomal RNA. We suggest that DNA gyrase-catalyzed alterations in DNA structure are involved in the regulation of the gene expressional program of outgrowing B. subtilis spores.

Bacillus subtilis↗
PubMedDate not supplied

Activity of ribosomal and tRNA promoters of Bacillus subtilis during sporulation.

The rrnB operon in Bacillus subtilis includes 21 tRNA genes downstream of the ribosomal genes. In addition to the dual promoters upstream of these ribosomal genes, a second promoter is found within the tRNA gene region. In this study, these two promoter regions were inserted before the lacZ gene and each was integrated as a single-copy into the B subtilis chromosome to examine their activity during sporulation. Both promoters exhibited similar strength and temporal downregulation from vegetative growth through t3 of sporulation. At t3, transcription from both promoters was approximately 20% of that in logarithmic growth. No obvious function of the second promoter is evident except to boost transcription of downstream genes. This function may be important because the ribosomal promoters of rrnB are weak relative to other ribosomal promoters. For instance, the rrnO promoters were much stronger than the rrnB ribosomal promoters and considerably more active at t3. Thus, all ribosomal promoters are not of the same strength nor are they transcriptionally downregulated to the same extent during sporulation. However, both the rRNA and tRNA promoters in the rrnB operon are similar in strength and downregulation.

Bacillus subtilis↗
PubMed1986

Synthesis of spoIIA and spoVA mRNA in Bacillus subtilis.

The expression of the spoIIA and spoVA sporulation loci of Bacillus subtilis was examined by using DNA-RNA hybridization to detect the time of appearance of their corresponding mRNA molecules in wild-type and asporogenous mutants of B. subtilis. From the size of the mRNA molecules it is clear that both the spoIIA and spoVA loci are polycistronic operons. Neither of the mRNA molecules is polyadenylated. The results also indicate the spoIIA operon is regulated by two promoters which become functional at different times.

Bacillus subtilis↗
PubMed2001

Expanding the genetic code of Escherichia coli.

A unique transfer RNA (tRNA)/aminoacyl-tRNA synthetase pair has been generated that expands the number of genetically encoded amino acids in Escherichia coli. When introduced into E. coli, this pair leads to the in vivo incorporation of the synthetic amino acid O-methyl-l-tyrosine into protein in response to an amber nonsense codon. The fidelity of translation is greater than 99%, as determined by analysis of dihydrofolate reductase containing the unnatural amino acid. This approach should provide a general method for increasing the genetic repertoire of living cells to include a variety of amino acids with novel structural, chemical, and physical properties not found in the common 20 amino acids.

Anticodon↗