Dimeric association of Escherichia coli RNA polymerase alpha subunits, studied by cleavage of single-cysteine alpha subunits conjugated to iron-(S)-1-[p-(bromoacetamido)benzyl]ethylenediaminetetraacetate.

Proximity relationships between the two associated monomers of the Escherichia coli RNA polymerase alpha subunit were studied using a set of four mutant alpha subunits, each with a single Cys residue at one of the naturally occurring positions (54, 131, 176, and 269). These mutant alpha subunits were conjugated with the cutting reagent iron-(S)-1-[p-(bromoacetamido)benzyl]ethylenediaminetetraacetate (Fe-BABE), and the peptide backbone was cleaved at locations near the modified Cys. Analysis of the cleavage sites identified segments within approximately 12 A of the conjugation site.

Adaptation of gene expression in stationary phase bacteria.

In nature, bacteria can survive for long periods in non-growing stationary states. Some species of bacteria survive by forming spores but non-spore-forming bacteria, including Escherichia coli, survive in the stationary phase. Gross changes in morphology and physiology occur in the stationary-phase bacteria and concomitantly a state of increased resistance against various stresses is established.

Evidence for transcription attenuation rendering cryptic a sigmaS-dependent promoter of the osmotically regulated proU operon of Salmonella typhimurium.

The osmotically regulated proU locus in Escherichia coli has two promoters, P1 and P2, that are recognized, respectively, by the sigmaS- and sigma70-bearing RNA polymerase holoenzymes. However, the equivalent of the P1 promoter does not appear to exist in Salmonella typhimurium. We demonstrate in this study that wild-type S.

[Transcription and replication of influenza virus genome].

The genome of influenza virus is composed of eight RNA segments of negative polarity. The RNA-dependent RNA polymerase is associated with each viral RNA (vRNA) segment and after infection, involved in both transcription (vRNA-directed synthesis of viral mRNA) and vRNA replication (vRNA-dependent synthesis of complementary RNA(cRNA) and cRNA-dependent synthesis of vRNA). The RNA polymerase is composed of three viral proteins, PB1, PB2 and PA.

Identification of two nucleotide-binding domains on the PB1 subunit of influenza virus RNA polymerase.

Influenza virus RNA polymerase is a multifunctional and multisubunit enzyme consisting of three viral P proteins, PB1, PB2, and PA. We have previously shown that radioactive 8-azido GTP (8-N3 GTP) was photo-crosslinked specifically to the PB1 subunit. Here we confirmed the specific crosslinking of PB1 using oxidized GTP and further identified the GTP analogue binding domains after proteolytic cleavage of the crosslinked PB1 with V8 protease.

RNA polymerase II subunits 2, 3, and 11 form a core subassembly with DNA binding activity.

RNA polymerase II purified from the fission yeast Schizosaccharomyces pombe consists of 10 species of subunit polypeptide. We introduced a histidine cluster tag sequence into the chromosomal rpb1 and rpb3 genes, which encode subunit 1 (Rpb1) and subunit 3 (Rpb3), respectively, and purified the RNA polymerase by Ni2+ affinity chromatography. After stepwise dissociation of the Rpb1- and Rpb3-tagged RNA polymerases fixed on Ni2+-resin by increasing concentrations of urea or guanidium hydrochloride, Rpb2-Rpb3-Rpb11 or Rpb2-Rpb3-Rpb11-Rpb10 complexes were obtained.

Repression and activation of promoter-bound RNA polymerase activity by Gal repressor.

By binding to the DNA site OE at position -60.5 in the gal operon, the GalR protein activates transcription from the P2 promoter located on the opposite face of DNA (position -5) and represses transcription from the P1 promoter located on the same face (position +1). GalR increases RNA polymerase binding at P2 and inhibits isomerization at P1 by forming a GalR-DNA-RNA polymerase ternary complex in each case.

Amino acid residues in the alpha-subunit C-terminal domain of Escherichia coli RNA polymerase involved in activation of transcription from the mtr promoter.

To examine the role of the amino acid residues (between positions 258 and 275 and positions 297 and 298) of the alpha-subunit of RNA polymerase in TyrR-mediated activation of the mtr promoter, we have carried out in vitro transcription experiments using a set of mutant RNA polymerases with a supercoiled mtr template. Decreases in factor-independent transcription in vitro by mutant RNA polymerases L262A, R265A, and K297A suggested the presence of a possible UP element associated with the mtr promoter. Mutational studies have revealed that an AT-rich sequence centered at -41 of the mtr promoter (SeqA) functions like an UP element.

Positioning of two alpha subunit carboxy-terminal domains of RNA polymerase at promoters by two transcription factors.

Interactions between the cAMP receptor protein (CRP) and the carboxy-terminal regulatory domain (CTD) of Escherichia coli RNA polymerase alpha subunit were analyzed at promoters carrying tandem DNA sites for CRP binding using a chemical nuclease covalently attached to alpha. Each CRP dimer was found to direct the positioning of one of the two alpha subunit CTDs. Thus, the function of RNA polymerase may be subject to regulation through protein-protein interactions between the two alpha subunits and two different species of transcription factors.

Gene organization and protein sequence of the small subunits of Schizosaccharomyces pombe RNA polymerase II.

RNA polymerase II purified from the fission yeast Schizosaccharomyces pombe contains 10 different species of polypeptides. Previously, we cloned and sequenced both cDNA and the genes encoding the four large subunits, Rpb1, Rpb2, Rpb3 and Rpb5. Later, other groups isolated the genes for Rpb6 and Rpb12 and cDNA for Rpb10.

Functional interaction of Escherichia coli RNA polymerase with inorganic polyphosphate.

Background: RNA polymerase from the stationary growth phase cells of Escherichia coli is tightly associated with an acidic compound(s) and exhibits altered promoter selectivity, with reduced transcriptional activity of the genes highly expressed in the exponentially growing cells. Here we have examined the nature of the RNA polymerase-associated acidic compound(s).

Results: RNA polymerase isolated from stationary-phase cells of E.

Promoter selectivity of Escherichia coli RNA polymerase sigmaF holoenzyme involved in transcription of flagellar and chemotaxis genes.

The rpoF gene of Escherichia coli codes for the RNA polymerase sigmaF (or sigma28) subunit, which is involved in transcription of the flagellar and chemotaxis genes. Both sigmaF and sigma70 (the major sigma subunit in growing cells) were overexpressed, purified to homogeneity, and compared with respect to activity and specificity. The affinity of sigmaF to core RNA polymerase (E) is higher than that of sigma70, as measured by gel filtration high-pressure liquid chromatography.

Identification of sigma factors for growth phase-related promoter selectivity of RNA polymerases from Streptomyces coelicolor A3(2).

We examined the promoter selectivity of RNA polymerase (RNAP) from Streptomyces coelicolor at two growth phases by in vitro transcription. Distinct sets of promoters were preferentially recognized by either exponential or stationary phase RNAP. No change in molecular weight or net charge of the core subunits was observed, suggesting that the associated specificity factors determined phase-specific promoter selectivity of the holoenzyme.

The molecular anatomy of influenza virus RNA polymerase.

The genome of influenza virus is composed of eight RNA segments of negative polarity. The RNA-dependent RNA polymerase is associated with each viral RNA (vRNA) segment and in virus-infected cells, involved in both transcription, i.e.

DNase I footprinting, DNA bending and in vitro transcription analyses of ClcR and CatR interactions with the clcABD promoter: evidence of a conserved transcriptional activation mechanism.

In Pseudomonas putida, benzoate and 3-chlorobenzoate are converted to catechol and 3-chlorocatechol, respectively, which are then catabolized to tricarboxylic acid cycle intermediates via the catBCA and clcABD pathways. The catBCA and clcABD operons are regulated by homologous transcriptional activators CatR and ClcR. Previous studies have demonstrated that in addition to sequence similarities, CatR and ClcR share functional similarities which allow catR to complement clcR.

Stimulatory effect of trehalose on formation and activity of Escherichia coli RNA polymerase E sigma38 holoenzyme.

The intracellular concentration of trehalose increases in the stationary-phase cells of Escherichia coli. The effects of trehalose on transcription in vitro by E. coli RNA polymerase were compared for two holoenzymes, E sigma70 and E sigma38, which were reconstituted from purified core enzyme and either sigma70 (the major sigma at the exponential growth phase) or sigma38 (the essential sigma at the stationary growth phase), respectively.

Flexible linker in the RNA polymerase alpha subunit facilitates the independent motion of the C-terminal activator contact domain.

The dynamic properties of the C-terminal one-third of the alpha subunit of RNA polymerase were investigated. The intact alpha subunit exhibited almost the same NMR spectral pattern as the isolated C-terminal fragment, indicating that the C-terminal domain retains the same conformation as the isolated fragment, and that its motion is independent of that of the associated N-terminal domain. Analysis of the NMR dynamics data for the intact alpha subunit indicated that at least 13 residues between the N and C-terminal domains show distinctly higher motional flexibility than the structured parts.

Physical mapping of a collection of Mael-generating amber mutations in the beta gene of Escherichia coli RNA polymerase and the functional effect of internal deletions constructed through their manipulation.

We report the fine mapping of 55 of our 95 amber mutations in the beta gene of Escherichia coli RNA polymerase by virtue of the unique MaeI restriction sites created by this subset of nonsense mutations (i.e. CTAG, where the amber codon is underlined).

Activation of the catBCA promoter: probing the interaction of CatR and RNA polymerase through in vitro transcription.

The soil bacterium Pseudomonas putida is capable of degrading many aromatic compounds, including benzoate, through catechol as an intermediate. The catabolism of catechol is mediated by the catBCA operon, whose induction requires the pathway intermediate cis,cis-muconate as an inducer and the regulatory protein, CatR. CatR also regulates the plasmid-borne pheBA operon of P.

The two alpha subunits of Escherichia coli RNA polymerase are asymmetrically arranged and contact different halves of the DNA upstream element.

RNA polymerase core enzyme of Escherichia coli is composed of two alpha subunits and one each of the beta and beta' subunits. The C-terminal domain of the RNA polymerase alpha subunit plays a key role in molecular communications with class I transcription factors and upstream (UP) elements of promoter DNA, using the same protein surface. To identify possible differences in the functional roles of the two alpha subunits, we have developed a reconstitution method for hybrid RNA polymerases containing two distinct alpha subunit derivatives in a defined orientation ("oriented alpha-heterodimer").

Nature of DNA binding and RNA polymerase interaction of the Bordetella pertussis BvgA transcriptional activator at the fha promoter.

The expression of virulence factor genes in Bordetella pertussis is mediated by the BvgA-BvgS two-component signal transduction system. The response regulator, BvgA, acts directly as a transcriptional activator at the loci encoding pertussis toxin (ptx) and filamentous hemagglutinin (fha). Previous studies have demonstrated that these two loci are differentially regulated by BvgA.

DNA flexibility of the UP element is a major determinant for transcriptional activation at the Escherichia coli acetate promoter.

The specific interaction of the upstream element-containing promoter of the Escherichia coli acetate operon with either the RNA polymerase holoenzyme or its alpha subunit has been analyzed by the base removal method. Our results indicate that: (i) direct and specific base contacts can be detected in the acetate promoter-alpha subunit complex; (ii) base elimination in the upstream element of the acetate promoter enhances the binding of RNA polymerase. A similar effect is observed when studying the interactions between RNA polymerase and the rrnB ribosomal operon P1 promoter.

Variation in RNA polymerase sigma subunit composition within different stocks of Escherichia coli W3110.

The composition of RNA polymerase sigma subunits was analyzed for stock strains of Escherichia coli K-12 W3110 in Japan. Heterogeneity was discovered with respect to two sigma subunits, sigma28 (sigmaF, the rpoF gene product) and sigma38 (sigmaS, the rpoS gene product). Five different types of W3110 were identified: A-type lineages have both sigma subunits in intact forms; B-type lineages carry a truncated sigma38 subunit and an intact sigma28 subunit; C-type lineages carry an intact sigma28 subunit but lack a sigma38 subunit; D-type lineages have only a sigma38 subunit without a sigma28 subunit; and E-type stocks lack both sigma subunits.

Synthesis of the protein cutting reagent iron (S)-1-(p-bromoacetamidobenzyl)ethylenediaminetetraacetate and conjugation to cysteine side chains.

Convenient methodology for preparation and conjugation of the protein-cutting iron chelate iron (S)-1-(p-bromoacetamidobenzyl) ethylenediaminetetraacetate (Fe-BABE) is given. This formulation of the reagent can be handled in a manner analogous to many other protein-labeling reagents, such as fluorescent probes or cross-linkers. By taking advantage of the recently discovered peptide hydrolysis reaction, the chelate may be tethered to a single site (e.

Transcription activation by the bacteriophage Mu Mor protein requires the C-terminal regions of both alpha and sigma70 subunits of Escherichia coli RNA polymerase.

Middle transcription of bacteriophage Mu requires Escherichia coli RNA polymerase and a Mu-encoded protein, Mor. Consistent with these requirements, the middle promoter, Pm, has a -10 hexamer but lacks a recognizable -35 hexamer. Interactions between Mor and RNA polymerase were studied using in vitro transcription, DNase I footprinting, and the yeast interaction trap system.