scholarly journals Interaction of sigma factor σN with Escherichia coli RNA polymerase core enzyme

2000 ◽  
Vol 352 (2) ◽  
pp. 539-547 ◽  
Author(s):  
David J. SCOTT ◽  
Anna L. FERGUSON ◽  
María-Trinidad GALLEGOS ◽  
Melinda PITT ◽  
Martin BUCK ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Sigma Factor ◽  
Analytical Ultracentrifugation ◽  
Spectroscopic Properties ◽  
Sedimentation Equilibrium ◽  
Equilibrium Binding ◽  
Kinetics Of

The equilibrium binding and kinetics of assembly of the DNA-dependent RNA polymerase (RNAP) σN-holoenzyme has been investigated using biosynthetically labelled 7-azatryptophyl- (7AW)σN. The spectroscopic properties of such 7AW proteins allows their absorbance and fluorescence to be monitored selectively, even in the presence of high concentrations of other tryptophan-containing proteins. The 7AWσN retained its biological activity in stimulating transcription from σN-specific promoters, and in in vitro gel electrophoresis assays of binding to core RNAP from Escherichia coli. Furthermore, five Trp → Ala single mutants of σN were shown to support growth under conditions of nitrogen limitation, and showed comparable efficiency in activating the σN-dependent nifH promoter in vivo, indicating that none of the tryptophan residues were essential for activity. The equilibrium binding of 7AWσN to core RNAP was examined by analytical ultracentrifugation. In sedimentation equilibrium experiments, absorbance data at 315nm (which reports selectively on the distribution of free and bound 7AWσN) established that a 1:1 complex was formed, with a dissociation constant lower than 2µM. The kinetics of the interaction between 7AWσN and core RNAP was investigated using stopped-flow spectrofluorimetry. A biphasic decrease in fluorescence intensity was observed when samples were excited at 280nm, whereas only the slower of the two phases was observed at 315nm. The kinetic data were analysed in terms of a mechanism in which a fast bimolecular association of σN with core RNAP is followed by a relatively slow isomerization step. The consequences of these findings on the competition between σN and the major sigma factor, σ70, in Escherichia coli are discussed.

scholarly journals Substitution of a Highly Conserved Histidine in the Escherichia coli Heat Shock Transcription Factor, σ32, Affects Promoter Utilization In Vitro and Leads to Overexpression of the Biofilm-Associated Flu Protein In Vivo

2007 ◽  
Vol 189 (23) ◽  
pp. 8430-8436 ◽  
Author(s):  
Olga V. Kourennaia ◽  
Pieter L. deHaseth
Keyword(s):  
Escherichia Coli ◽  
Heat Shock ◽  
Rna Polymerase ◽  
Sigma Factor ◽  
Stable Complex ◽  
Tryptophan Residue ◽  
Specific Sequence ◽  
Bacterial Rna

ABSTRACT The heat shock sigma factor (σ32 in Escherichia coli) directs the bacterial RNA polymerase to promoters of a specific sequence to form a stable complex, competent to initiate transcription of genes whose products mitigate the effects of exposure of the cell to high temperatures. The histidine at position 107 of σ32 is at the homologous position of a tryptophan residue at position 433 of the main sigma factor of E. coli, σ70. This tryptophan is essential for the strand separation step leading to the formation of the initiation-competent RNA polymerase-promoter complex. The heat shock sigma factors of all gammaproteobacteria sequenced have a histidine at this position, while in the alpha- and deltaproteobacteria, it is a tryptophan. In vitro the alanine-for-histidine substitution at position 107 (H107A) destabilizes complexes between the GroE promoter and RNA polymerase containing σ32, implying that H107 plays a role in formation or maintenance of the strand-separated complex. In vivo, the H107A substitution in σ32 impedes recovery from heat shock (exposure to 42°C), and it also leads to overexpression at lower temperatures (30°C) of the Flu protein, which is associated with biofilm formation.

scholarly journals The FecI Extracytoplasmic-Function Sigma Factor of Escherichia coli Interacts with the β′ Subunit of RNA Polymerase

2003 ◽  
Vol 185 (6) ◽  
pp. 1796-1802 ◽  
Author(s):  
Susanne Mahren ◽  
Volkmar Braun
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Sigma Factor ◽  
Nitrilotriacetic Acid ◽  
Ferric Citrate ◽  
Β Subunit ◽  
Ecf Sigma Factor ◽  
K 12

ABSTRACT Transcription of the ferric citrate transport system of Escherichia coli K-12 is mediated by the extracytoplasmic-function (ECF) sigma factor FecI, which is activated by ferric citrate in the growth medium. By using a bacterial two-hybrid system, it was shown in vivo that FecI binds to the β′ subunit of RNA polymerase. The inactive mutant protein FecI(K155E) displayed reduced binding to β′, and small deletions along the entire FecI protein led to total impairment of β′ binding. In vitro, FecI was retained on Ni2+-nitrilotriacetic acid agarose loaded with a His-tagged β′1-313 fragment and coeluted with β′1-313. Binding of FecI to β′ and β′1-313 was enhanced by FecR1-85, which represents the cytoplasmic portion of the FecR protein that transmits the inducing signal across the cytoplasmic membrane. Interaction of FecR with FecI was demonstrated by showing that isolated FecR inhibited degradation of FecI by trypsin. This is the first demonstration of binding of an ECF sigma factor of the FecI type to the β′ subunit of RNA polymerase and of binding being enhanced by the protein that activates the ECF sigma factor.

scholarly journals Effects of distortions by A-tracts of promoter B-DNA spacer region on the kinetics of open complex formation by Escherichia coli RNA polymerase.

2003 ◽  
Vol 50 (4) ◽  
pp. 909-920 ◽  
Author(s):  
Iwona K Kolasa ◽  
Tomasz Łoziński ◽  
Kazimierz L Wierzchowski
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Transcription Initiation ◽  
Specific Interaction ◽  
Structural Data ◽  
Structural Morphology ◽  
Promoter Dna ◽  
Sigma Subunit ◽  
Kinetics Of

A-tracts in DNA due to their structural morphology distinctly different from the canonical B-DNA form play an important role in specific recognition of bacterial upstream promoter elements by the carboxyl terminal domain of RNA polymerase alpha subunit and, in turn, in the process of transcription initiation. They are only rarely found in the spacer promoter regions separating the -35 and -10 recognition hexamers. At present, the nature of the protein-DNA contacts formed between RNA polymerase and promoter DNA in transcription initiation can only be inferred from low resolution structural data and mutational and crosslinking experiments. To probe these contacts further, we constructed derivatives of a model Pa promoter bearing in the spacer region one or two An (n = 5 or 6) tracts, in phase with the DNA helical repeat, and studied the effects of thereby induced perturbation of promoter DNA structure on the kinetics of open complex (RPo) formation in vitro by Escherichia coli RNA polymerase. We found that the overall second-order rate constant ka of RPo formation, relative to that at the control promoter, was strongly reduced by one to two orders of magnitude only when the A-tracts were located in the nontemplate strand. A particularly strong 30-fold down effect on ka was exerted by nontemplate A-tracts in the -10 extended promoter region, where an involvement of nontemplate TG (-14, -15) sequence in a specific interaction with region 3 of sigma-subunit is postulated. A-tracts in the latter location caused also 3-fold slower isomerization of the first closed transcription complex into the intermediate one that precedes formation of RPo, and led to two-fold faster dissociation of the latter. All these findings are discussed in relation to recent structural and kinetic models of RPo formation.

Alteration of adipocyte calcium homeostasis by Escherichia coli endotoxin

1985 ◽  
Vol 248 (3) ◽  
pp. R331-R338
Author(s):  
K. M. Nelson ◽  
J. A. Spitzer
Keyword(s):  
Escherichia Coli ◽  
Cell Surface ◽  
Calcium Homeostasis ◽  
Compartmental Analysis ◽  
In Vitro Exposure ◽  
Exchange Kinetics ◽  
Intracellular Binding ◽  
Kinetics Of

The present study evaluated calcium homeostasis in rat adipocytes after either in vivo or in vitro exposure to Escherichia coli endotoxin. Fat cells from endotoxin-treated rats showed an enhanced uptake of 45Ca. In an attempt to differentiate between 45Ca binding to the cell surface and intracellular 45Ca accumulation, adipocytes were exposed to 5 mM LaCl3. The amount of 45Ca remaining associated with lanthanum-treated adipocytes was taken to be located intracellularly and was increased in adipocytes from endotoxin-treated rats. The amount of 45Ca displaced by lanthanum was also increased in adipocytes from endotoxin-treated rats. This suggested that the endotoxin-induced increase of 45Ca accumulation included both cell surface and intracellular binding sites. Compartmental analysis of the exchange kinetics of cell-associated 45Ca with 40Ca in the medium indicated a 77% increase in the size of the cell surface compartment of adipocytes from endotoxin-treated rats compared with controls. In addition, endotoxin treatment altered the flux of calcium from the cells to the medium. In vitro exposure of freshly prepared adipocytes to 250 or 750 micrograms endotoxin/ml did not produce a perturbation of adipocyte calcium homeostasis. The results indicate that endotoxin induces alterations in the ability of adipocytes to regulate calcium translocations, suggesting that some metabolic and hormonal aspects of endotoxins' actions may be mediated through perturbation of cellular calcium homeostasis.

Application of Escherichia coli phage K1E DNA-dependent RNA polymerase for in vitro RNA synthesis and in vivo protein production in Bacillus megaterium

2010 ◽  
Vol 88 (2) ◽  
pp. 529-539 ◽  
Author(s):  
Simon Stammen ◽  
Franziska Schuller ◽  
Sylvia Dietrich ◽  
Martin Gamer ◽  
Rebekka Biedendieck ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Bacillus Megaterium ◽  
Protein Production ◽  
Rna Synthesis ◽  
Escherichia Coli Phage

scholarly journals In vivo and in vitro phosphorylation of DNA-dependent RNA polymerase of Escherichia coli by bacteriophage-T7-induced protein kinase.

1975 ◽  
Vol 72 (7) ◽  
pp. 2506-2510 ◽  
Author(s):  
W. Zillig ◽  
H. Fujiki ◽  
W. Blum ◽  
D. Janekovic ◽  
M. Schweiger ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Protein Kinase ◽  
Rna Polymerase ◽  
Bacteriophage T7

scholarly journals Sigma Factor F Does Not Prevent Rifampin Inhibition of RNA Polymerase or Cause Rifampin Tolerance in Mycobacterium tuberculosis

2010 ◽  
Vol 192 (20) ◽  
pp. 5472-5479 ◽  
Author(s):  
Ruben C. Hartkoorn ◽  
Claudia Sala ◽  
Sophie J. Magnet ◽  
Jeffrey M. Chen ◽  
Florence Pojer ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Rna Polymerase ◽  
Stationary Phase ◽  
Sigma Factor ◽  
Potent Inhibitor ◽  
Sigma Factors ◽  
Antituberculosis Drugs ◽  
Axenic Cultures

ABSTRACT The tolerance of Mycobacterium tuberculosis to antituberculosis drugs is a major reason for the lengthy therapy needed to treat a tuberculosis infection. Rifampin is a potent inhibitor of RNA polymerase (RNAP) in vivo but has been shown to be less effective against stationary-phase bacteria. Sigma factor F is associated with bacteria entering stationary phase and has been proposed to impact rifampin activity. Here we investigate whether RNAP containing SigF is more resistant to rifampin inhibition in vitro and whether overexpression of sigF renders M. tuberculosis more tolerant to rifampin. Real-time and radiometric in vitro transcription assays revealed that rifampin equally inhibits transcription by RNAP containing sigma factors SigA and SigF, therefore ruling out the hypothesis that SigF may be responsible for increased resistance of the enzyme to rifampin in vitro. In addition, overexpression or deletion of sigF did not alter rifampin susceptibility in axenic cultures of M. tuberculosis, indicating that SigF does not affect rifampin tolerance in vivo.

scholarly journals ςBldN, an Extracytoplasmic Function RNA Polymerase Sigma Factor Required for Aerial Mycelium Formation in Streptomyces coelicolor A3(2)

2000 ◽  
Vol 182 (16) ◽  
pp. 4606-4616 ◽  
Author(s):  
Maureen J. Bibb ◽  
Virginie Molle ◽  
Mark J. Buttner
Keyword(s):  
Rna Polymerase ◽  
Sigma Factor ◽  
Streptomyces Coelicolor ◽  
Aerial Mycelium ◽  
Sigma Factors ◽  
Colony Development ◽  
Null Mutant ◽  
Wild Type

ABSTRACT Sporulation mutants of Streptomyces coelicolor appear white because they are defective in the synthesis of the gray polyketide spore pigment, and such white (whi) mutants have been used to define 13 sporulation loci. whiN, one of five new whi loci identified in a recent screen of NTG (N-methyl-N′-nitro-N-nitrosoguanidine)-inducedwhi strains (N. J. Ryding et al., J. Bacteriol. 181:5419–5425, 1999), was defined by two mutants, R112 and R650. R650 produced frequent spores that were longer than those of the wild type. In contrast, R112 produced long, straight, undifferentiated hyphae, although rare spore chains were observed, sometimes showing highly irregular septum placement. Subcloning and sequencing showed thatwhiN encodes a member of the extracytoplasmic function subfamily of RNA polymerase sigma factors and that the sigma factor has an unusual N-terminal extension of approximately 86 residues that is not present in other sigma factors. A constructed whiN null mutant failed to form aerial mycelium (the “bald” phenotype) and, as a consequence, whiN was renamed bldN. This observation was not totally unexpected because, on some media, the R112 point mutant produced substantially less aerial mycelium than its parent, M145. The bldN null mutant did not fit simply into the extracellular signaling cascade proposed for S. coelicolor bld mutants. Expression of bldN was analyzed during colony development in wild-type and aerial mycelium-deficientbld strains. bldN was transcribed from a single promoter, bldNp. bldN transcription was developmentally regulated, commencing approximately at the time of aerial mycelium formation, and depended on bldG and bldH, but not on bldA, bldB, bldC,bldF, bldK, or bldJ or onbldN itself. Transcription from the p1 promoter of the response-regulator gene bldM depended onbldN in vivo, and the bldMp1 promoter was shown to be a direct biochemical target for ςBldN holoenzyme in vitro.

scholarly journals Cra-Dependent Transcriptional Activation of theicd Gene of Escherichia coli

1999 ◽  
Vol 181 (3) ◽  
pp. 893-898 ◽  
Author(s):  
Jean-François Prost ◽  
Didier Nègre ◽  
Christelle Oudot ◽  
Katsuhiko Murakami ◽  
Akira Ishihama ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Transcriptional Activation ◽  
Α Subunit ◽  
P2 Promoter ◽  
Catabolite Repressor ◽  
A Site ◽  
Extension Analysis

ABSTRACT The icd gene of Escherichia coli, encoding isocitrate dehydrogenase, was shown to be expressed from two different promoters: the previously identified icd P1 and a newly detected second promoter, icd P2, whose expression is positively regulated by the catabolite repressor-activator protein Cra, formerly called FruR. In each case, we determined the mRNA start site by primer extension analysis of in vivo transcripts and examined the interaction of the icd control region with either RNA polymerase or Cra. We observed that (i) the Cra factor binds to and activates transcription from a site centered at position −76.5 within the icd P2 promoter region and (ii) three particular mutations in the C-terminal end of the α subunit of RNA polymerase (L262A, R265A, and N268A) considerably diminish transcription initiating from the icd P2 promoter, as shown by in vitro experiments performed in the presence of mutant RNA polymerases carrying Ala substitutions.

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