Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.7.6 (RNA polymerase)
34,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Production of the main virulence determinant pectate lyases (Pels) of the phytopathogenic bacterium Erwinia chrysanthemi is modulated by a complex regulatory network involving the repressor proteins KdgR, PecS and PecT and the activator systems Pir, ExpI-ExpR and CRP. Of these regulators, CRP and PecT are particularly important since the absence of CRP or a slight overproduction of PecT leads to a drastic reduction in synthesis of Pel species. Recently, it has been shown that production of Pel species is strongly reduced in an E. chrysanthemi hns mutant, suggesting an activator function of the nucleoid-associated protein H-NS in the expression of the pel genes. Here, we report that the reduced synthesis of Pel species in the hns mutant results from a negative control, exerted by H-NS, on the transcription of the regulatory gene pecT. This H-NS/PecT cascade regulation is one of the first elucidations of a positive effect of H-NS on target gene expression. Moreover, we found that H-NS also represses the expression of expI, expR and pel genes. H-NS control is the result of H-NS binding to extended regions within the pecT, expI, expR and pel genes. Investigation of the simultaneous binding of CRP, RNA polymerase (RNAP) and H-NS on the pelD gene revealed that these three proteins form a nucleoprotein com-plex. Together, these data indicate that, by exerting a negative control at multiple levels, H-NS plays a crucial role in the E. chrysanthemi pel regulatory network.
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PMID:H-NS-dependent activation of pectate lyases synthesis in the phytopathogenic bacterium Erwinia chrysanthemi is mediated by the PecT repressor. 1192 28

Site-directed mutagenesis was conducted in the regulatory region of the Escherichia coli udp gene at promoter sites responsible for binding regulatory proteins CRP and CytR as well as RNA polymerase (the core-promoter containing the--10 sequence). In mutants with an "improved"--10 region, a partial relief from the control of the cAMP-CRP transcription activation complex occurred, and the negative CytR repressor regulation was reduced. In contrast, mutant promoters with a weak Pribnow block or with a deletion that completely eliminates the core-promoter exhibited an increased ability to titrate the CytR protein in vivo. On the other hand, the affinity of CytR for DNA in mutants with an altered--10 region was the same as in the wild-type udp promoter. After introduction of mutations affecting binding sites for CRP (CRP1 and CRP2), the negative effect of the CytR protein on promoter transcription was fully abolished. The CRP1 binding site was shown to play the main role in the activation of the promoter by the cAMP-CRP complex, whereas the CRP2 site participates in the formation of the repressor complex. Mutations in the main and additional CytR binding sites were isolated and characterized. On the basis of these data, it is concluded that the modification of each structural element of the udp regulatory region (binding sites for CytR, CRP, or RNA polymerase) caused changes in the overall pattern of the promoter regulation.
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PMID:[Functional interrelationship between elements of the Escherichia coli udp gene promotor responsible for binding regulatory proteins CytR, CRP, and RNA polymerase]. 1239 83

The interaction between CRP, T127L, S128A, and CRP and RNA polymerase bound to a 104 bp synthetic promoter were determined by ITC at 298 K and ranges from a deltaG(b) degrees = 1.4 +/- 0.8 kJ mol(-)(1) (cAMP-ligated S128A) to 4.5 +/- 0.3 kJ mol(-)(1) (cAMP-ligated double mutant CRP) with endothermicities that range from 4 +/- 3 kJ mol(-)(1) (cAMP-ligated CRP) to 47 +/- 8 kJ mol(-)(1) (cGMP-ligated T127L). The interaction is, thus, entropically driven, exhibits enthalpy-entropy compensation, and increases the binding affinity of the RNA polymerase to the promoter by factors ranging from 1.7 +/- 0.1 (cAMP-ligated S128A) to 6.1 +/- 0.1 (cAMP-ligated CRP). Although the binding affinities to the promoter alone, except for cAMP-ligated S128A, are the same as to a shorter 40 bp duplex containing the same CRP consensus binding site sequence (conDNA), the binding enthalpies of CRP/mutant to the promoter are lower by factors of 2-3 x than the corresponding binding enthalpies to conDNA. Small angle neutron scattering measurements on the DNA-CRP/mutant complexes in D(2)O/H(2)O solutions exhibit an increase in the Rg of the CRP/mutant component from 22 to 27-31 A that can be attributed to a conformational change in the N-terminal domain of CRP. The Rg = 27 A for the bound conDNA can be attributed to a slight unwinding of the DNA in solution that would also enhance the activation of transcription. The Rg = 53 +/- 3 A for the bound promoter is attributed to bending of the promoter in solution that can be responsible for the lower CRP/mutant-promoter binding endothermicities.
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PMID:Entropic nature of the interaction between promoter bound CRP mutants and RNA polymerase. 1259 May 82

The protein array methodology is used to study DNA-protein and protein-protein interactions governing gene expression from the Bacillus stearothermophilus PargCo promoter-operator region. Using probes labelled with near-infrared fluorescence dyes with exitation characteristics close to 700 or 800 nm, it is possible to detect signals from proteins (purified or non-purified in Escherichia coli cell extracts) immobilised on a nitrocellulose membrane with a high sensitivity (almost 12 amol of a spotted protein for protein-DNA interactions). Protein array data are confirmed by other methods indicating that molecular interactions of the order 10(-7) M can be monitored with the proposed protein array approach. We show that the PargCo region is a target for binding at least three types of regulatory proteins, ArgR repressors from thermophilic bacteria, the E. coli RNA polymerase alpha subunit and cyclic AMP binding protein CRP. We also demonstrate that the high strength of the PargC promoter is related to an upstream element that binds to the E. coli RNA polymerase alpha subunit.
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PMID:Dissecting DNA-protein and protein-protein interactions involved in bacterial transcriptional regulation by a sensitive protein array method combining a near-infrared fluorescence detection. 1274 44

The Escherichia coli FNR protein is a global transcription regulator that activates gene expression via interactions with the RNA polymerase alpha subunit C-terminal domain. Using preparations of E. coli RNA polymerase holoenzyme, specifically labelled with a DNA cleavage reagent, we have determined the location and orientation of the C-terminal domain of the RNA polymerase alpha subunit in transcriptionally competent complexes at a class II FNR-dependent promoter. We conclude that one alpha subunit C-terminal domain binds immediately upstream of FNR, and that its position and orientation is the same as at similar promoters dependent on CRP, another E. coli transcription activator that is related to FNR. In complementary experiments, we show that the second alpha subunit C-terminal domain of RNA polymerase can be repositioned by upstream-bound CRP, but not by upstream-bound FNR.
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PMID:Location of the Escherichia coli RNA polymerase alpha subunit C-terminal domain at an FNR-dependent promoter: analysis using an artificial nuclease. 1475 8

The mechanism of isomerization (basepair openings) during transcription initiation by RNA polymerase at the galP1 promoter of Escherichia coli was investigated by 2-aminopurine (2,AP) fluorescence. The fluorescence of 2,AP is quenched in DNA duplex and enhanced when the basepair is distorted or deformed. The increase of 2,AP fluorescence was used to monitor basepair distortion at several individual positions in the promoter. We observed that basepair distortions during isomerization are a multi-step process. Three distinct hitherto unresolved steps in kinetic terms were observed, where significant fluorescence change occurs: a fast step with a half-life of around 1 s, which is followed by two slower steps occurring with a half-life in the range of minutes at 25 degrees C. Contrary to commonly held expectations, basepairs at different positions opened by 2,AP assays without any obvious pattern, suggesting that basepair opening is an asynchronous multi-step process. cAMP.CRP, which activates transcription at galP1, enhanced the rate-limiting step.
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PMID:Asynchronous basepair openings in transcription initiation: CRP enhances the rate-limiting step. 1496 88

Effect of mutations in the -10 and -35 regions of the udp gene promoter on the nature of its regulation by CytR and CRP proteins was studied. In studies of expression of mutant promoters, competition between RNA polymerase and the CytR repressor for the promoter region of the udp gene was shown. In the presence of the improved -10 region, the introduction of a substitution 15C-->T (that is the presence of the elongated Pribnow block) resulted in the CRP-independent transcription of the udp gene promoter. The binding site CRP2 was shown to be indispensable for the maximum promoter activation by the transcription-activating cAMP-CRP complex. Both positive (cAMP-CRP complex) and negative (CytR) regulation of the promoter was virtually fully abolished after the introduction of mutations leading to the creation of canonical sequences in -10 and -35 promoter regions.
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PMID:[Structural-functional analysis of the promoter region of Escherichia coli udp gene]. 1502 96

The transcriptional activator, MarA, interacts with RNA polymerase (RNAP) to activate promoters of the mar regulon. Here, we identify the interacting surfaces of MarA and of the carboxy-terminal domain of the alpha subunit of RNAP (alpha-CTD) by NMR-based chemical shift mapping. Spectral changes were monitored for a MarA-DNA complex upon titration with alpha-CTD, and for alpha-CTD upon titration with MarA-DNA. The mapping results were confirmed by mutational studies and retention chromatography. A model of the ternary complex shows that alpha-CTD uses a '265-like determinant' to contact MarA at a surface distant from the DNA. This is unlike the interaction of alpha-CTD with the CRP or Fis activators where the '265 determinant' contacts DNA while another surface of the same alpha-CTD molecule contacts the activator. These results reveal a new versatility for alpha-CTD in transcriptional activation.
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PMID:Versatility of the carboxy-terminal domain of the alpha subunit of RNA polymerase in transcriptional activation: use of the DNA contact site as a protein contact site for MarA. 1545 4

To investigate the determining factors in the selection of the transcription start points (tsp) by RNA polymerase of Escherichia coli, we systematically deleted or substituted single base pairs (bps) at 25 putative critical positions in the two extended -10 promoters, P1 and P2, of the gal operon. These changes extend downstream from -24 to +1 of the P1 promoter. In vitro transcription assays using supercoiled DNA templates revealed a preference for a purine in the non-template strand for tsp in both promoters. The optimal tsp is the 11th bp counting downstream from the -10 position. A single bp deletion anywhere from -10 to +1 switched the tsp to the next available purine 2-3 bp downstream on the non-template strand whereas deleting a single bp at position from -24 to -11 did not affect the tsp. The nature of the 10 bp sequence of the -10 to -1 region, while affecting promoter strength, did not influence tsp. The cAMP-CRP complex, which stimulates P1 and represses P2, did not affect the tsp selection process. The rules of tsp selection by RNA polymerase containing sigma70 in gal and pyr promoters discussed here may be applicable to others.
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PMID:Axiom of determining transcription start points by RNA polymerase in Escherichia coli. 1549 60

Transcription initiation is a major target for the regulation of gene expression in all organisms. Transcription activators can stimulate different steps in the initiation process including the initial binding of RNA polymerase (RNAP) to the promoter and a subsequent promoter-melting step. Typically, kinetic assays are required to determine whether an activator exerts its effect on the initial binding of RNAP or on the promoter-melting step. Here we take advantage of a mutant Escherichia coli RNAP that is deficient in promoter melting to assess the ability of an activator to stabilize the initial binding of RNAP to the promoter. For the well-characterized activator CRP, we show that this RNAP mutant can be used to distinguish between effects on initial binding and promoter melting; these results provide an independent confirmation of the results of kinetic analysis. We then employ the melting-deficient RNAP mutant to demonstrate an effect of an artificial activator of transcription on the initial binding of RNAP. Our findings demonstrate that a melting-deficient RNAP mutant can be used to trap a normally unstable intermediate in transcription initiation, thus providing a novel tool for probing activation mechanism.
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PMID:An RNA polymerase mutant deficient in DNA melting facilitates study of activation mechanism: application to an artificial activator of transcription. 1549 4


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