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)

The tdc operon is subject to CRP-controlled catabolite repression. Expression of the operon is also induced anaerobically, although this regulation does not rely on direct control by either FNR or ArcA. Recently, the anaerobic expression of the tdc operon was found to be fortuitously induced in the presence of glucose by a heterologous gene isolated from the Gram-positive anaerobe Clostridium butyricum. The gene, termed tcbC, encoded a histone-like protein of 14.5 kDa. Using tdc-lacZ fusions, it was shown that TcbC did not activate tdc expression by functionally replacing any of the operon regulators. In vitro transcription analyses with RNA polymerase and CRP revealed that faithful CRP-dependent transcription initiation occurred only on supercoiled templates. No specific, CRP-dependent transcription initiation was observed on relaxed or linear DNA templates. Surprisingly, purified His-tagged TcbC activated transcription from a relaxed, circular template, but not from supercoiled or linear templates. Examination of the CRP binding site of the tdc promoter revealed that it was located 43.5 bp upstream of the transcription initiation site. Repositioning of the CRP site at -41.5 bp abolished activation by the TcbC protein and allowed CRP-dependent transcription to occur on linear, relaxed and supercoiled templates. TcbC bound DNA non-specifically; however, in topoisomerase I relaxation assays, it was demonstrated that TcbC imposed torsional constraints on negatively supercoiled DNA, which influenced the ability of the enzyme to relax the topoisomers. Taken together, these results strongly suggest that TcbC activates transcription of tdc by altering the local topological status of the tdc promoter and that, in the wild-type tdc promoter, the CRP binding site is misaligned to allow transcription to occur only under optimal conditions. Indeed, in vivo transcription analyses revealed that repositioning of the CRP binding site to -41.5 bp resulted in high-level, CRP-dependent transcription, even under catabolite-repressing conditions, and that transcription was no longer influenced by TcbC. Remarkably, however, anaerobic regulation of the mutant promoter was retained. This indicates that the other tdc regulators, TdcA and TdcR, govern anaerobic transcription activation by CRP.
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PMID:A novel mechanism controls anaerobic and catabolite regulation of the Escherichia coli tdc operon. 1125 44

The development of genome sequencing and DNA microarray analysis of gene expression gives rise to the demand for data-mining tools. BioProspector, a C program using a Gibbs sampling strategy, examines the upstream region of genes in the same gene expression pattern group and looks for regulatory sequence motifs. BioProspector uses zero to third-order Markov background models whose parameters are either given by the user or estimated from a specified sequence file. The significance of each motif found is judged based on a motif score distribution estimated by a Monte Carlo method. In addition, BioProspector modifies the motif model used in the earlier Gibbs samplers to allow for the modeling of gapped motifs and motifs with palindromic patterns. All these modifications greatly improve the performance of the program. Although testing and development are still in progress, the program has shown preliminary success in finding the binding motifs for Saccharomyces cerevisiae RAP1, Bacillus subtilis RNA polymerase, and Escherichia coli CRP. We are currently working on combining BioProspector with a clustering program to explore gene expression networks and regulatory mechanisms.
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PMID:BioProspector: discovering conserved DNA motifs in upstream regulatory regions of co-expressed genes. 1126 34

Mlc is a global regulator of carbohydrate metabolism. Recent studies have revealed that Mlc is depressed by protein-protein interaction with enzyme IICB(Glc), a glucose-specific permease, which is encoded by ptsG. The mlc gene has been previously known to be transcribed by two promoters, P1(+1) and P2(+13), and have a binding site of its own gene product at +16. However, the mechanism of transcriptional regulation of the gene has not yet been established. In vitro transcription assays of the mlc gene showed that P2 promoter could be recognized by RNA polymerase containing the heat shock sigma factor final sigma(32) (E sigma(32)) as well as E sigma(70), while P1 promoter is only recognized by E sigma(70). The cyclic AMP receptor protein and cyclic AMP complex (CRP.cAMP) increased expression from P2 but showed negative effect on transcription from P1 by E sigma(70), although it had little effect on transcription from P2 by E sigma(32) in vitro. Purified Mlc repressed transcription from both promoters, but with different degrees of inhibition. In vivo transcription assays using wild type and mlc strains indicated that the level of mlc expression was modulated less than 2-fold by glucose in the medium with concerted action of CRP.cAMP and Mlc. A dramatic increase in mlc expression was observed upon heat shock or in cells overexpressing final sigma(32), confirming that E sigma(32) is involved in the expression of mlc. Induction of ptsG P1 and pts P0 transcription by glucose was also dependent on E sigma(32). These results indicate that E sigma(32) plays an important role in balancing the relative concentration of Mlc and EIICB(Glc) in response to availability of glucose in order to maintain inducibility of the Mlc regulon at high growth temperature.
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PMID:Heat shock RNA polymerase (E sigma(32)) is involved in the transcription of mlc and crucial for induction of the Mlc regulon by glucose in Escherichia coli. 1134 70

Expression of the microcin C51 operon in Escherichia coli cells is regulated as a function of the phase of growth; it is stimulated during the decelerating phase of growth. Using single-copy P(mcc)-lac transcriptional fusion (the promoter region of the microcin C51 operon fused to a promoterless lac operon in lambda phage), we showed that transcription from the microcin operon promoter is dependent on sigma(s) (RpoS) factor. However, some level of P(mcc)-lac expression is possible in rpoS null mutants, indicating that another sigma factor might be involved in transcription of the microcin C51 operon. Overproduction of sigma70 decreased Pmcc-directed transcription, presumably as a result of competition of sigma factors for the limited amount of core RNA polymerase. The cyclic AMP-CRP complex was shown to stimulate transcription from Pmcc: the absence of CRP or cAMP in crp or cya mutant cells strongly decreased the level of P(mcc)-lac expression. The production of C51 microcin decreased or was absent in rpoS, crp and cya mutant cells. Leucine-responsive protein Lrp and histone-like protein H-NS repressed P(mcc)-lac expression in the exponential and decelerating phases of growth. In studies of P(mcc)-lac expression in double mutant cells, we showed that proteins CRP, Lrp and H-NS acted in rpoS-dependent and rpoS-independent ways in transcription of the microcin C51 operon. Mutation hns(-) resulted in an increase in P(mcc)-lac expression in crp, rpoS and lrp mutant cells, as in wild-type cells.
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PMID:Regulation of microcin C51 operon expression: the role of global regulators of transcription. 1144 15

CooA is a CO-sensing protein that activates the transcription of genes encoding the CO-oxidation (coo) regulon, whose polypeptide products are required for utilizing CO as an energy source in Rhodospirillum rubrum. CooA binds to a position overlapping the -35 element of the P(cooF) promoter, similar to the arrangement of class II CRP (cAMP receptor protein)- and FNR (fumarate and nitrate reductase activator protein)-dependent promoters when expressed in Escherichia coli. Gain-of-function CooA variants were isolated in E. coli following mutagenesis of the portion of cooA encoding the effector-binding domain. Some of the mutations affect regions of CooA that are homologous to the activating regions (AR2 and AR3) previously identified in CRP and FNR, whereas others affect residues that lie in a region of CooA between AR2 and AR3. These CooA variants are comparable to wild-type (WT) CooA in DNA binding affinity in response to CO but differ in transcription activation, presumably because of altered interactions with E. coli RNA polymerase. Based on predictions of similarity to CRP and FNR, loss-of-function CooA variants were obtained in the AR2 and AR3 regions that have minimal transcriptional activity, yet have WT-like DNA binding affinities in response to CO. This study demonstrates that WT CooA contains AR2- and AR3-like surfaces that are required for optimal transcription activation.
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PMID:Mapping CooA.RNA polymerase interactions. Identification of activating regions 2 and 3 in CooA, the co-sensing transcriptional activator. 1152 88

Transcription initiation by the stress-associated sigma(S)-containing RNA polymerase holoenzyme (E sigma(S)) in Escherichia coli is often subject to complex regulation that involves multiple additional regulators and histone-like proteins. csiD is a stationary phase-inducible sigma(S)-dependent gene in E. coli that requires activation by cAMP-CRP (bound to a site centred at -68.5 nucleotides upstream of the transcriptional start site) and is positively modulated by the abundant nucleoid-associated proteins H-NS and Lrp. By shifting the CRP box to positions between -80.5 and -60.5, we could demonstrate that: (i) activation is equally helix phase dependent as at classic class I promoters; (ii) E sigma(S) prefers a CRP box location at -68.5/-70.5, whereas E sigma(70) is nearly inactive with such an arrangement; and (iii) with the CRP site moved to -60.5, transcription can be initiated efficiently by both holoenzymes. The csiD promoter region also contains a distal UP-element half-site located downstream of the CRP box, as demonstrated by mutational studies, in which this element was either eliminated or completed to a full UP-element. The UP-element half-site favours E sigma(S)-mediated expression, whereas with the full UP-element, nearly wild-type levels of csiD transcription were observed in the absence of sigma(S). Finally, we show that the two histone-like proteins, H-NS and Lrp, both act by influencing activation by cAMP-CRP, but do so by different mechanisms. In particular, H-NS directly or indirectly increases positional stringency for the CRP binding site. The implications of these findings with respect to sigma factor selectivity, activation mechanisms used by the two holoenzymes and the architecture of sigma(S)-dependent promoters are discussed.
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PMID:Role of activator site position and a distal UP-element half-site for sigma factor selectivity at a CRP/H-NS-activated sigma(s)-dependent promoter in Escherichia coli. 1153 38

In the deoP2 promoter of Escherichia coli, a transcription activator, cAMP-CRP, binds at two sites, centered at -41.5 and -93.5 from the start site of transcription, while a repressor, CytR, binds to a space between the two cAMP-CRP complexes. The mechanisms for the cAMP-CRP-mediated transcription activation and CytR-mediated transcription repression were investigated in vitro using purified components. We classified the deoP2 promoter as a class II cAMP-CRP-dependent promoter, primarily by the action of cAMP-CRP at the downstream site. Interestingly, we also found that deoP2 carries an "UP-element" immediately upstream of the downstream cAMP-CRP site. The UP-element overlaps with the DNA site for CytR. However, it was observed that CytR functions with the RNA polymerase devoid of the C-terminal domain of the alpha-subunit as well as with intact RNA polymerase. The mechanism of repression by CytR proposed in this study is that the cAMP-CRP bound at -41.5 undergoes an allosteric change upon direct interaction with CytR such that it no longer maintains a productive interaction with the N-terminal domain of alpha, but instead acts as a repressor to interfere with RNA polymerase acting on deoP2.
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PMID:Repression of deoP2 in Escherichia coli by CytR: conversion of a transcription activator into a repressor. 1157 71

The Escherichia coli melAB promoter is co-dependent upon two transcription activators, MelR and the cyclic AMP receptor protein, CRP. In this study we demonstrate positive co-operativity between the binding of MelR and CRP at the melAB promoter, which provides a simple mechanism for its co-dependence. MelR binds to four sites, centred at positions -42.5, -62.5, -100.5 and -120.5 relative to the melAB transcription start point. When MelR is pre-bound, CRP is able to bind to a target located between MelR at positions -62.5 and -100.5. This increases the occupation of the two downstream sites for MelR, which is essential for transcription activation. We have identified residues within activating region 1 (AR1) of CRP that are important in transcription activation of the melAB promoter. At simple CRP-dependent promoters, the surface of CRP containing these residues is involved in contacting the RNA polymerase alpha subunit. Our results show that, at the melAB promoter, the surface of CRP containing AR1 contacts MelR rather than RNA polymerase. Thus, MelR and CRP activate transcription by a novel mechanism in which they bind co-operatively to adjacent sites and form a bacterial enhanceosome.
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PMID:A simple mechanism for co-dependence on two activators at an Escherichia coli promoter. 1174 92

The hpt gene, which encodes hypoxanthine phosphoribosyltransferase, is located next to, but transcribed in the opposite direction to, the gcd gene, which codes for a membrane-bound glucose dehydrogenase, at 3.1 min on the Escherichia coli genome. In their promoter-operator region, putative regulatory elements for integration host factor (IHF) and for the complex comprising 3', 5'-cyclic AMP (cAMP) and its receptor protein (CRP) are present, and they overlap the promoters for hpt and gcd, respectively. The involvement of IHF and cAMP-CRP, as well as the corresponding putative cis-acting elements, in the expression of the two genes was investigated by using lacZ operon fusions. In an adenylate cyclase-deficient strain, addition of cAMP increased the expression of hpt and reduced the expression of gcd. In agreement with this observation, the introduction of mutations into the putative binding element for the cAMP-CRP complex enhanced the expression of gcd. In contrast, mutations introduced into the putative IHF-binding elements increased the level of hpt expression. Similar results were obtained with IHF-defective strains. Thus, the expression of the two genes is regulated in a mutually exclusive manner. Additional experiments with mutations at the -10 sequence of the gcd promoter suggest that the binding of RNA polymerase to the hpt promoter interferes with the interaction of RNA polymerase with the gcd promoter, and vice versa.
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PMID:Differential control by IHF and cAMP of two oppositely oriented genes, hpt and gcd, in Escherichia coli: significance of their partially overlapping regulatory elements. 1181 Feb 62

Fis is a versatile transactivator that functions at many different promoters. Fis activates transcription at the RpoS-dependent proP P2 promoter when bound to a site that overlaps the minus sign35 hexamer by a mechanism that requires the C-terminal domain of the alpha subunit of RNA polymerase (alphaCTD). The region on Fis responsible for activating transcription through the alphaCTD has been localized to a short beta-turn near the DNA-binding determinant on one subunit of the Fis homodimer. We report here that Fis-dependent activation of proP P2 transcription requires two discrete regions on the alphaCTD. One region, consisting of residues 264-265 and 296-297, mediates DNA binding. A second patch, comprising amino acid residues 271-273, forms a ridge on the surface of the alphaCTD that we propose interacts with Fis. The accompanying paper shows that these same regions on alphaCTD are utilized for transcriptional activation at the rrnB and rrnE P1 promoters by Fis bound to a site upstream of the core promoter (centered at minus sign71/minus sign72). In addition to stimulation of proP P2 transcription by Fis, CRP co-activates this promoter when bound to a remote site upstream from the promoter (centered at -121.5). RNA polymerase preparations lacking one alphaCTD of the alpha dimer were employed to demonstrate that the beta'-associated alpha(II)CTD was utilized preferentially by Fis at proP P2 in the presence and absence of CRP. These experiments define the overall architecture of the proP P2 initiation complex where Fis and CRP each function through a different alphaCTD.
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PMID:The C-terminal domains of the RNA polymerase alpha subunits: contact site with Fis and localization during co-activation with CRP at the Escherichia coli proP P2 promoter. 1186 15


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