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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)

Transcription of the proP gene, encoding a transporter of the osmoprotectants proline and glycine betaine, is controlled from two promoters, P1 and P2, that respond primarily to osmotic and stationary-phase signals, respectively. The P1 promoter is normally expressed at a very low level under low or normal medium osmolarity. We demonstrate that the binding of the cyclic AMP (cAMP) receptor protein (CRP) to a site centered at -34.5 within the promoter is responsible for the low promoter activity under these conditions. A brief period of reduced CRP binding in early log phase corresponds to a transient burst of P1 transcription upon resumption of growth in Luria-Bertani broth. A CRP binding-site mutation or the absence of a functional crp gene leads to high constitutive expression of P1. We show that the binding of CRP-cAMP inhibits transcription by purified RNA polymerase in vitro at P1, but this repression is relieved at moderately high potassium glutamate concentrations. Likewise, open-complex formation at P1 in vivo is inhibited by the presence of CRP under low-osmolarity conditions. Because P1 expression can be further induced by osmotic upshifts in a delta crp strain or in the presence of the CRP binding-site mutation, additional controls exist to osmotically regulate P1 expression.
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PMID:Cyclic AMP receptor protein functions as a repressor of the osmotically inducible promoter proP P1 in Escherichia coli. 907 29

In CytR regulated promoters in Escherichia coli, the cAMP-CRP complex acts as a transcriptional activator as well as a co-repressor for the CytR protein. Repression by CytR depends on the formation of nucleoprotein complexes in which CytR binds cooperatively to the DNA with one or two cAMP-CRP complexes. Here, we demonstrate that in order to establish CytR regulation in a cAMP-CRP dependent class II promoter with a single CRP site (CRP site centred around position -40.5) in which the CytR operator is located upstream of the CRP site, high affinity binding sites for both regulators are required. The efficiency of CytR regulation was observed to be modulated by RNA polymerase (RNAP)-promoter interactions. Specifically, in class II promoters with a single CRP site, the efficiency of CytR regulation was found to correlate inversely with cAMP-CRP independent promoter activity. These observations can be reconciled in a competition model for CytR regulation in which CytR and RNAP compete for cooperative binding with cAMP-CRP to the promoters in vivo. In this model, two mutually exclusive ternary complexes can be formed: a CytR/cAMP-CRP/promoter repression complex and an RNAP/cAMP-CRP/promoter activation complex. Thus, CytR regulation critically depends on formation of a repression complex that binds the promoter with sufficiently high affinity to exclude formation of the competing activation complex. We suggest that the transition from repression to activation involves a switch in the protein-protein interactions made by cAMP-CRP from CytR to RNAP. On the basis of the regulatory features of the promoters analysed here, we speculate about the advantages offered by the structural complexity of natural CytR/cAMP-CRP regulated promoters.
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PMID:Design of CytR regulated, cAMP-CRP dependent class II promoters in Escherichia coli: RNA polymerase-promoter interactions modulate the efficiency of CytR repression. 908 66

A library of random mutations in the Escherichia coli fnr gene has been screened to identify positive control mutants of FNR that are defective in transcription activation at Class I promoters. Single amino acid substitutions at D43, R72, S73, T118, M120, F181, F186, S187 and F191 identify a surface of FNR that is essential for activation which, presumably, makes contact with the C-terminal domain of the RNA polymerase alpha subunit. This surface is larger than the corresponding activating surface of the related transcription activator, CRP. To identify the contact surface in the C-terminal domain of the RNA polymerase alpha subunit, a library of mutations in the rpoA gene was screened for alpha mutants that interfered with transcription activation at Class I FNR-dependent promoters. Activation was reduced by deletions of the alpha C-terminal domain, by substitutions known to affect DNA binding by alpha, by substitutions at E261 and by substitutions at L300, E302, D305, A308, G315 and R317 that appear to identify contact surfaces of alpha that are likely to make contact with FNR at Class I promoters. Again, this surface differs from the surface used by CRP at Class I CRP-dependent promoters.
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PMID:Transcription activation at class I FNR-dependent promoters: identification of the activating surface of FNR and the corresponding contact site in the C-terminal domain of the RNA polymerase alpha subunit. 932 53

Escherichia coli contains two differentially regulated aconitase genes, acnA and acnB. Two acnA promoters transcribing from start points located 407 bp (P1acnA) and 50 bp (P2acnA) upstream of the acnA coding region, and one acnB promoter (PacnB) with a start point 95 bp upstream of the acnB coding region, were identified by primer extension analysis. A 2.8 kb acnA monocistronic transcript was detected by Northern blot hybridization, but only in redox-stressed (methyl-viologen-treated) cultures, and a 2.5 kb acnB monocistronic transcript was detected in exponential- but not stationary-phase cultures. These findings are consistent with previous observations that acnA is specifically subject to SoxRS-mediated activation, whereas acnB encodes the major aconitase that is synthesized earlier in the growth cycle than AcnA. Further studies with acn-lacZ gene fusions and a wider range of transcription regulators indicated that acnA expression is initiated by sigma 38 from P1acnA, and from P2acnA it is activated directly or indirectly by CRP, FruR, Fur and SoxRS, and repressed by ArcA and FNR. In contrast, acnB expression is activated by CRP and repressed by ArcA, FruR and Fis from PacnB. Comparable studies with fum-lacZ fusions indicated that transcription of fumC, but not of fumA or fumB, is initiated by RNA polymerase containing sigma 38. It is concluded that AcnB is the major citric acid cycle enzyme, whereas AcnA is an aerobic stationary-phase enzyme that is specifically induced by iron and redox-stress.
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PMID:Transcriptional regulation of the aconitase genes (acnA and acnB) of Escherichia coli. 942 4

During transcription initiation at bacterial promoters, the C-terminal domain of the RNA polymerase alpha subunit (alphaCTD) can interact with DNA-sequence elements (known as UP elements) and with activator proteins. We have constructed a series of semi-synthetic promoters carrying both an UP element and a consensus DNA-binding site for the Escherichia coli cAMP receptor protein (CRP; a factor that activates transcription by making direct contacts with alphaCTD). At these promoters, the UP element was located at a variety of distances upstream of the CRP-binding site, which was fixed at position -41.5 bp upstream of the transcript start. At some positions, the UP element caused enhanced promoter activity whereas, at other positions, it had very little effect. In no case was the CRP-dependence of the promoter relieved. DNase I and hydroxyl-radical footprinting were used to study ternary RNA polymerase-CRP-promoter complexes formed at two of the most active of these promoters, and co-operativity between the binding of CRP and purified alpha subunits was studied. The footprints show that alphaCTD binds to the UP element as it is displaced upstream but that this displacement does not prevent alphaCTD from being contacted by CRP. Models to account for this are discussed.
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PMID:Spacing requirements for interactions between the C-terminal domain of the alpha subunit of Escherichia coli RNA polymerase and the cAMP receptor protein. 946 38

The general stress-induced sigma subunit sigma s of Escherichia coli RNA polymerase is closely related to the vegetative sigma factor sigma 70. In view of their very similar promoter specificity in vitro, it is unclear how sigma factor selectivity in the expression of sigma s-dependent genes is generated in vivo. The csiD gene is such a strongly sigma s-dependent gene. In contrast to sigma s, which is induced in response to many different stresses, csiD, whose expression is driven from a single promoter, is induced by carbon starvation only. To our knowledge, the csiD promoter is the first characterized promoter which is not only exclusively dependent on sigma s-containing RNA polymerase (E sigma s), but also requires an activator, cAMP-CRP. In addition, leucine-responsive regulatory protein (Lrp) acts as a positive modulator of csiD expression. Also in vitro, E sigma s is more efficient than E sigma 70 in csiD promoter binding, open complex formation and run-off transcription, which might be due to the poor match of the csiD -35 region to the sigma 70 consensus and to transcription by E sigma s being less dependent on contacts in this region. By DNase I protection experiments, a cAMP-CRP binding site centered at -68.5 nucleotides upstream of the csiD transcriptional start site was identified. While cAMP-CRP stimulates E sigma 70 binding, it does not promote open complex formation by E sigma 70, but does so in conjunction with E sigma s. With linear templates, cAMP-CRP significantly stimulates E sigma s-mediated in vitro transcription, whereas transcription by E sigma 70 is negligible and hardly stimulated by cAMP-CRP. These findings may reflect different or less stringent positional requirements for an activator site for E sigma s than for E sigma 70, and indicate that cAMP-CRP contributes to sigma factor selectivity at the csiD promoter. In vitro transcription experiments with super-coiled templates, however, revealed significant cAMP-CRP-stimulated transcription also by E sigma 70. Yet, under these conditions, H-NS was found to restore E sigma s specificity by strongly interfering with cAMP-CRP/E sigma 70-dependent transcription. Lrp strongly and cooperatively binds to multiple sites located between positions -14 and -102 (in a way that suggests DNA wrapping around multiple Lrp molecules) and moderately stimulates in vitro transcription, especially with E sigma s. In summary, we conclude that the csiD promoter has an intrinsic preference for E sigma s, but that also protein factors such as cAMP-CRP, Lrp and probably H-NS as well as DNA conformation contribute to its strong E sigma s selectivity. Furthermore, this strong E sigma s preference in combination with a requirement for high concentrations of the essential activator cAMP-CRP ensures csiD expression under conditions of carbon starvation, but not other stress conditions.
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PMID:Molecular analysis of the regulation of csiD, a carbon starvation-inducible gene in Escherichia coli that is exclusively dependent on sigma s and requires activation by cAMP-CRP. 951 7

During transcription activation at FNR-dependent promoters where the DNA site for FNR overlaps the -35 element, a surface-exposed activating region in the upstream subunit of the FNR dimer interacts with the C-terminal domain of the RNA polymerase alpha subunit. Starting with a cloned fnr gene encoding a defective FNR derivative carrying substitutions in this activating region, we screened a library of random mutations to identify substitutions that restored FNR activity. Activity can be restored by substitutions at residues T118, E47 and K60. The locations of these residues identify three separate surface-exposed regions of FNR that can play a role in transcription activation. These three regions appear to be analogues of Activating Region 1, Activating Region 2 and Activating Region 3 of the cyclic AMP receptor protein, CRP: our results underscore the similarities between FNR and CRP.
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PMID:Transcription activation by Escherichia coli FNR protein: similarities to, and differences from, the CRP paradigm. 954 62

Glucose stimulates the expression of ptsG encoding the major glucose transporter in Escherichia coli. We isolated Tn 10 insertion mutations that confer constitutive expression of ptsG. The mutated gene was identified as mlc, encoding a protein that is known to be a repressor for transcription of several genes involved in carbohydrate utilization. Expression of ptsG was eliminated in a mlc crp double-negative mutant. The Mlc protein was overproduced and purified. In vitro transcription studies demonstrated that transcription of ptsG is stimulated by CRP-cAMP and repressed by Mlc. The action of Mlc is dominant over that of CRP-cAMP. DNase I footprinting experiments revealed that CRP-cAMP binds at two sites centred at -40.5 and -95.5 and that Mlc binds at two regions centred around -8 and -175. The binding of CRP-cAMP stimulated the binding of RNA polymerase to the promoter while Mlc inhibited the binding of RNA polymerase but not the binding of CRP-cAMP. Gel-mobility shift assay indicated that glucose does not affect the Mlc binding to the ptsG promoter. Our results suggest that Mlc is responsible for the repression of ptsG transcription and that glucose modulates the Mlc activity by unknown mechanism.
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PMID:A global repressor (Mlc) is involved in glucose induction of the ptsG gene encoding major glucose transporter in Escherichia coli. 978 86

The sigma subunit of RNA polymerase orchestrates basal transcription by first binding to core RNA polymerase and then recognizing promoters. Using a series of 16 alanine-substitution mutations, we show that residues in a narrow region of Escherichia coli sigma70 (590 to 603) are involved in transcription activation by a mutationally altered CRP derivative, FNR and AraC. Homology modeling of region 4 of sigma70 to the closely related NarL or 434 Cro proteins, suggests that the five basic residues implicated in activation are either in the C terminus of a long recognition helix that includes residues recognizing the -35 hexamer region of the promoter, or in the subsequent loop, and are ideally positioned to permit interaction with activators. The only substitution that has a significant effect on activator-independent transcription is at R603, indicating that this residue of sigma70 may play a distinct role in transcription initiation.
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PMID:Identification of a contact site for different transcription activators in region 4 of the Escherichia coli RNA polymerase sigma70 subunit. 987 55

The lacP DNA binding and activation characteristics of CRP having amino acid substitutions at position 127 were investigated. Wild-type (WT) and T127C CRP footprinted lacP DNA in the presence of DNase I in a cAMP-dependent manner. The T127G, T127I, and T127S forms of CRP failed to footprint lacP both in the absence and in the presence of cAMP. Consistent with these data, WT and T127C CRP:cAMP complexes exhibited high affinity for the lacP CRP site whereas T127G, T127I, or T127S CRP:cAMP complexes exhibited low affinity for the lacP CRP site. CRP:cAMP:RNA polymerase (RNAP) complexes formed at lacP in reactions that contained WT, T127C, T127G, T127I, or T127S CRP. These results demonstrate that allosteric changes important for cAMP-mediated CRP activation are differentially affected by amino acid substitution at position 127. Proper cAMP-mediated reorientation of the DNA binding helices required either threonine or cysteine at position 127. However, cAMP-dependent interaction of CRP with RNAP was accomplished regardless of the amino acid at position 127. RNAP:lacP complexes that supported high-level lac RNA synthesis formed rapidly in reactions that contained WT or T127C CRP whereas RNAP:lacP complexes that supported only low-level lac RNA synthesis formed at slower rates in reactions that contained T127I or T127S CRP. The T127G CRP:cAMP:RNAP:lacP complex failed to activate lacP. The results of this study lead us to conclude that threonine 127 plays an important role in transduction of the signal from the CRP cyclic nucleotide binding pocket that promotes proper orientation of the DNA binding helices and only a minor, if any, role in the functional exposure of the CRP RNAP interaction domain.
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PMID:Position 127 amino acid substitutions affect the formation of CRP:cAMP:lacP complexes but not CRP:cAMP:RNA polymerase complexes at lacP. 1032 Mar 51


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