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 simultaneous binding of Gal repressor (GalR), catabolite activator protein (CAP or CRP), and RNA polymerase (RNAP) to the promoter region of the Escherichia coli gal operon has been analyzed thermodynamically, by quantitative DNase I "footprint" titration analysis, and structurally, by the use of hydroxyl radical (.OH) and 5-phenylphenanthroline (5OPP) "footprinting." In the absence of regulatory proteins, the preference of RNAP for one (P1) of the two gal operon overlapping promoters (P1 and P2) is -0.4 +/- 0.2 kcal/mol, indicating only a small energetic preference for P1. The simultaneous binding of CAP and RNAP occurs with 10-fold cooperativity, with greater than 99% of the CAP-RNAP complex present at the P1 promoter. This cooperativity is inhibited by the binding of GalR to the upstream operator, OE, but does not result in the repartitioning of RNAP between the P1 and P2 promoters. These results suggest that the CAP-RNAP cooperativity and promoter partitioning are not linked and are consistent with a mechanism by which GalR binding to OE represses transcription by inhibiting the CAP-RNAP cooperativity. It is suggested that the CAP-RNAP cooperativity is dependent upon contacts made by the complex with the upstream DNA and that GalR binding to OE prevents these contacts from occurring. Changes in nuclease reactivity at the internal operator OI (centered at +53.5) take place upon RNAP binding. These changes are dependent on the DNA sequence present at OI and on the presence or absence of CAP. They are independent of the helical phasing between the promoters and OI and of the distance between them. These results suggest that RNAP can directly communicate with events occurring at both the external and the internal operator sequences without direct contact between repressor molecules bound at their cognate sites.
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PMID:Interactions between RNA polymerase and the positive and negative regulators of transcription at the Escherichia coli gal operon. 861 94

The kinetics of open complex formation were measured by migration retardation assay and DNase I footprinting at the activator-dependent promoters ara P1, lac P1 and gal P1. In each case, the rate of open complex formation was significantly faster if the activator, AraC for ara and CAP for lac and gal, had been added before RNA polymerase. The results indicate that complexes of transcriptional activators, RNA polymerase and promoter can exist in two states, one which can form open complexes rapidly and one which cannot.
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PMID:Obligatory activator-polymerase addition order at promoters. 869 98

FIS, a site-specific DNA binding and bending protein, is a global regulator of gene expression in Escherichia coli. The ribosomal RNA promoter rrnB P1 is activated 3- to 7-fold in vivo by a FIS dimer that binds a DNA site immediately upstream of the DNA binding site for the C-terminal domain (CTD) of the alpha subunit of RNA polymerase (RNAP). In this report, we identify several FIS side chains important specifically for activation of transcription at rrnB P1. These side chains map to positions 68, 71 and 74, in and flanking a surface-exposed loop adjacent to the helix-turn-helix DNA binding motif of the protein. We also present evidence suggesting that FIS activates transcription at rrnB P1 by interacting with the RNAP alphaCTD. Our results suggest a model for FIS-mediated activation of transcription at rrnB P1 that involves interactions between FIS and the RNAP alphaCTD near the DNA surface. Although FIS and the transcription activator protein CAP have little structural similarity, they both bend DNA, use a similarly disposed activation loop and target the same region of the RNAP alphaCTD, suggesting that this is a common architecture at bacterial promoters.
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PMID:Molecular anatomy of a transcription activation patch: FIS-RNA polymerase interactions at the Escherichia coli rrnB P1 promoter. 900 76

Transcription activation at Class II CAP-dependent promoters provides a paradigm for understanding how a single activator molecule can make multiple interactions with the transcription machinery, with each interaction being responsible for a specific mechanistic consequence. At Class II CAP-dependent promoters, the DNA target site for CAP is centred near position -42, overlapping and replacing the -35 determinant for binding of RNA polymerase (RNAP). Transcription activation requires two distinct mechanistic components. The first component is 'anti-inhibition,' overcoming an inhibitory effect of the RNAP alpha subunit C-terminal domain (alpha CTD). This component involves direct contact between amino acids 156-164 (activating region 1) of the upstream subunit of the CAP dimer and a target in alpha CTD. The second component is 'direct activation', facilitating isomerization of the RNAP-promoter closed complex to the transcriptionally competent open complex. This component involves direct contact between amino acids 19, 21 and 101 (activating region 2) of the downstream subunit of the CAP dimer and a target in the RNAP alpha subunit N-terminal domain (alpha NTD).
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PMID:Transcription activation at class II CAP-dependent promoters. 907 23

Bacteriophage P2 late transcription requires the product of the P2 ogr gene. Ogr-dependent transcription from P2 late promoters is blocked by certain point mutations affecting the alpha subunits of the host RNA polymerase. An alanine scan spanning the putative activation target in the alpha C-terminal domain (alphaCTD) was carried out to identify individual residues essential for Ogr-dependent transcription from P2 late promoters. In addition, the effects of alanine substitutions in the regions of the alphaCTD previously reported to affect CAP-dependent activation of the lac promoter and UP-element DNA binding were examined. Residues E286, V287, L289 and L290 in helix 3, and residue L300 at the beginning of helix 4, define a surface-exposed patch on the alphaCTD important for Ogr-dependent activation. These residues, adjacent to the recently identified DNA-binding determinants, constitute a newly identified activation surface for protein:protein contact. Alanine substitutions at some of the residues that affect UP-element DNA binding also impaired activation. This suggests that upstream DNA-alpha contacts, in addition to alpha-Ogr contacts, may be important in P2 late transcription. Other residues implicated in the interaction of alpha with CAP are not required for activation by Ogr, consistent with previous genetic evidence suggesting that these activators contact different sites on the alphaCTD.
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PMID:Activation of P2 late transcription by P2 Ogr protein requires a discrete contact site on the C terminus of the alpha subunit of Escherichia coli RNA polymerase. 939 9

Repression of the divergent nagE - B operons requires NagC binding to two operators which overlap the nagE and nagB promoters, resulting in formation of a DNA loop. Binding of the cAMP/CAP activator to its site, adjacent to the nagE operator, stabilizes the DNA loop in vitro. The DNA of the nagE-B intergenic region is intrinsically bent, with the bend centred on the CAP site. We show that displacement of the CAP site by 6 bp results in complete derepression of the two operons. This derepression is observed even in the absence of cAMP/CAP binding and despite the fact that the two NagC operators are still in phase, demonstrating that the inherently bent structure of the DNA loop is important for repression. Since no interaction between NagC and CAP has been detected, we propose that the role of CAP in the repression loop is architectural, stabilizing the intrinsic bend. The cAMP/CAP complex is necessary for activation of the nagE-B promoters. In this case protein-protein contacts between CAP and RNA polymerase are necessary for full activation, but at least a part of the activation is likely due to an effect of CAP binding altering DNA structure.
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PMID:DNA bending and expression of the divergent nagE-B operons. 946 34

The Escherichia coli bgl promoter is kept in a repressed state by silencer sequences which flank the promoter and by the histone-like protein H-NS. Silencing of the bgl promoter is likely due to the formation of a repressing nucleoprotein complex of which H-NS is an essential component. Here, we show that silencing is abolished by the binding of Lac or lambda repressors to their respective operators that were inserted within the bgl upstream silencer. Efficient activation of bgl operon transcription by Lac and lambda repressors was independent of the position and phasing of the operators with respect to the promoter. Activation by Lac and lambda repressors as shown here is unprecedented. We conclude that the activation of bgl transcription by both repressors is achieved by a novel mechanism, that is by alteration of the repressing nucleoprotein complex rather than by protein-protein interactions with RNA polymerase and the catabolite activator protein, CAP.
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PMID:Lac and lambda repressors relieve silencing of the Escherichia coli bgl promoter. Activation by alteration of a repressing nucleoprotein complex. 983 11

Activation of promoters by multiple transcription factors might occur through favorable contacts of the activators with themselves or RNA polymerase, or by changes in DNA geometry that enhance formation of the transcription complex. Transcription of the Escherichia coli uhpT gene, encoding the organophosphate transporter, requires the response regulator UhpA and is stimulated by the global regulator protein CAP. CAP binds to the uhpT promoter at a single site, centered at -103.5 bp relative to the start of transcription, and UhpA binds to multiple sites between positions -80 and -32. Overexpression of UhpA did not reduce the degree of CAP stimulation of uhpT-lacZ expression, showing that CAP action is more complex than enhancement of the binding of UhpA. Footprinting experiments demonstrated that UhpA and CAP modestly stimulated each other's binding to the uhpT promoter, but did not affect the positioning of the binding sites. An in vitro transcription system was used to examine the contribution of each transcription factor at the uhpT promoter. Action of UhpA and CAP proteins was not affected by template supercoiling. Kinetic analyses of productive and abortive initiation showed that CAP acted both to stabilize by fivefold the open promoter complexes formed in the presence of UhpA and to enhance by twofold the rate of their formation. These results indicate that open complex formation requires UhpA and that CAP stabilizes the open complex.
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PMID:Separate contributions of UhpA and CAP to activation of transcription of the uhpT promoter of Escherichia coli. 1051 97

The wild-type Escherichia coli bgl promoter is silent in vivo but active in vitro. Silencing in vivo is directed by silencer sequences that flank the promoter, and requires nucleoid-associated protein H-NS and other unidentified cellular factors. Here we show that the DNA bending protein FIS is a repressor of the bgl promoter. Two FIS binding sites, centred at positions -52 and -27, overlap the CAP binding site and the -35 box respectively. FIS efficiently competes with CAP for binding to the wild-type promoter. However, FIS does not prevent binding of RNA polymerase. It interferes with the formation of a heparin-resistant complex and represses transcription initiation up to 40-fold. The presence of CAP has very little effect on the FIS-mediated repression of the wild-type bgl promoter in vitro. However, when a bgl promoter allele was tested that carries an improved CAP binding site (which leads to activation in vivo) CAP effectively counteracted repression by FIS in vitro. These results suggest that FIS contributes to silencing of the wild-type bgl promoter in vivo, presumably in the early exponential phase when FIS is predominantly expressed.
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PMID:Antagonistic control of the Escherichia coli bgl promoter by FIS and CAP in vitro. 1076 Jan 65

Using overlapping and mutant oligonucleotides as probes, gel mobility assays and competition experiments identified a sequence from -47 to -32 bp upstream of the LIM2 CAP site, which a lens protein complex bound with high affinity which appeared to bind only to the "sense" strand of the double-stranded DNA molecule. This sequence consisted of a string of four guanine residues followed by seven other nucleotides (AACCTAA) and followed by another four guanines, i.e. GGGGAACCTAAGGGG, called the Hsu element. Promoter-CAT constructs containing this sequence or mutations of the sequence indicated that the Hsu element is located within the basal promoter, and is essential for expression of the LIM2 gene. The trans factors binding to the Hsu element are present throughout development, and appear to be lens-specific. Since the LIM2 gene promoter does not contain a classic TATA box, the Hsu element may serve as the site for binding the RNA polymerase complex.
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PMID:Identification of a lens-specific cis-acting element within the basal promoter of the human lens intrinsic membrane protein MP19 gene (LIM2). 1596 79


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