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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 protein sigma 54 associates with Escherichia coli core RNA polymerase to form a holoenzyme that binds promoters but is inactive in the absence of enhancer activation. Here, mutants of sigma 54 enabled polymerases to transcribe without enhancer protein and adenosine triphosphate. The mutations are in leucines within the NH2-terminal glutamine-rich domain of sigma 54. Multiple leucine substitutions mimicked the effect of enhancer protein, which suggests that the enhancer protein functions to disrupt a leucine patch. The results indicate that sigma 54 acts both as an inhibitor of polymerase activity and as a receptor that interacts with enhancer protein to overcome this inhibition, and that these two activities jointly confer enhancer responsiveness.
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PMID:Converting Escherichia coli RNA polymerase into an enhancer-responsive enzyme: role of an NH2-terminal leucine patch in sigma 54. 748 5

sigma 54 is a rare bacterial protein that substitutes for sigma 70 in the case of Escherichia coli genes transcribed by certain activators with enhancer protein-like properties. It contains a strongly acidic region of previously unknown function. Gel mobility-shift assays using sigma 54 deletion mutants show that this region is essential for sigma 54 to bind the core RNA polymerase and recruit it to the promoter. Multiple-point mutational analysis shows that the acidic amino acids and overlapping periodic hydrophobic amino acids are necessary for this binding. Related sequences are not found within the core binding determinant of sigma 70, which binds the same core RNA polymerase. This comparison suggests that the core RNA polymerase interacts differently with the two sigma factors, likely contributing to the critical differences in transcription mechanism in the two cases.
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PMID:RNA polymerase binding using a strongly acidic hydrophobic-repeat region of sigma 54. 813 58

Sigma 54 is a required factor for bacterial RNA polymerase to respond to enhancers and directs a mechanism that is a hybrid between bacterial and eukaryotic transcription. Three pathways were found that bypass the enhancer requirement in vitro. These rely on either deletion of the sigma 54 N terminus or destruction of the DNA consensus -12 promoter recognition element or altering solution conditions to favor transient DNA melting. Each of these allows unstable heparin-sensitive pre-initiation complexes to form that can be driven to transcribe in the absence of both enhancer protein and ATP beta-gamma hydrolysis. These disparate pathways are proposed to have a common basis in that multiple N-terminal contacts may mediate the interactions between the polymerase and the DNA region where melting originates. The results raise possibilities for common features of open complex formation by different RNA polymerases.
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PMID:Multiple pathways to bypass the enhancer requirement of sigma 54 RNA polymerase: roles for DNA and protein determinants. 927 58

The double strand binding protein A (DsbA) of bacteriophage T4 is one of several viral gene products participating in transcriptional regulation. These proteins interact or associate with the host RNA polymerase core enzyme, enabling the enzyme to successively initiate transcription at different classes of viral promoters: early, middle and late. This leads to a temporally controlled expression of the T4 gene products. The DsbA binding site overlaps the late promoter region, and DsbA binding seems to intensify transcription of late genes in vitro, possibly acting as an enhancer protein (Molecular Biology of Phage T4, Karam, 1994). To further investigate the function and structure of DsbA, we overexpressed the protein in E. coli and purified it to homogeneity. Physiological functionality of the recombinant protein was shown by gel retardation experiments and by circular dichroism (CD) spectroscopy. DsbA shows strong bands in the near UV-CD spectra. The far UV-CD spectroscopy analysis shows alpha-helices to be the main secondary structure elements. This is in agreement with secondary structure predictions. A possible helix-turn-helix motif in the center of the protein could be identified. Results from crosslinking and sedimentation analyses show that DsbA forms a dimer in solution. The thermal unfolding curve fits a dimer-two-state-folding-model, and the unfolding temperature was concentration dependent. Therefore, dimerization should supply the main portion of the free energy of stabilization of deltaG0 = 42 kJ/mol.
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PMID:Overexpression and structural characterization of the phage T4 protein DsbA. 950 17

Results of binding assays using DNA fork junction probes indicate that sigma 54 contains multiple determinants that regulate melting to allow RNA polymerase to remain in closed promoter complexes in order to respond to enhancers. Gel mobility shift studies indicate that the -12 promoter element and parts of sigma 54 act together to form a molecular switch that controls melting. The DNA sequences and the sigma 54 N-terminus help direct polymerase to the location within the -12 promoter element where melting will initiate. However, the fork junction that would lead to melting does not form, due to the action of an inhibitory DNA element. Such unregulated melting is inhibited further by the lack of availability of the single-strand binding elements, which are needed to spread opening from the junction to the transcription start site. Thus, in the absence of looping enhancer protein, proper regulation is maintained as the sigma 54 polymerase remains bound in an inactive state. These complex protein-DNA interactions allow the controls over protein recruitment and DNA melting to be separated, enhancing the diversity of accessible mechanisms of transcription regulation.
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PMID:A fork junction DNA-protein switch that controls promoter melting by the bacterial enhancer-dependent sigma factor. 1039 88

Transcription control at the melting step is not yet understood. Here, band shift, cross-linking, and transcription experiments on diverse DNA probes were used with two bacterial RNA polymerase holoenzymes that differ in how they regulate melting. Data indicated that both sigma(54) and sigma(70) holoenzymes assume a default closed form that cannot establish single-strand binding. Upon activation the enzymes are converted to an open form that can bind simultaneously to the upstream fork junction and to the melted transcription start site. The key difference is that sigma(54) imposes tighter regulation by creating a complex molecular switch at -12/-11; the current data show that this switch can be thrown by activator. In this case an ATP-bound enhancer protein causes sigma(54) to alter its cross-linking pattern near -11 and also causes a reorganization of holoenzyme: DNA interactions, detected by electrophoretic mobility-shift assay. At a temperature-dependent sigma(70) promoter, elevated temperature alone can assist in triggering conformational changes that enhance the engagement of single-strand DNA. Thus, the two sigma factors modify the same intrinsic opening pathway to create quite different mechanisms of transcriptional regulation.
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PMID:Promoter opening by sigma(54) and sigma(70) RNA polymerases: sigma factor-directed alterations in the mechanism and tightness of control. 1097 Aug 87

The NtrC-family PhhR protein of Pseudomonas putida is involved in the control of the metabolism of aromatic amino acids, and it is a dual regulatory protein. When PhhR acts as an activator, it stimulates transcription from its cognate promoters with RNA polymerase/sigma(70) rather than with sigma(54), as is the case for most members of the family. The target binding sites in repressed and activated promoters are defined by the 5'-TGTAAAN(6)TTTACA-3' consensus sequence. PhhR binds to target sites as a dimer with affinity in the range of 0.03 to 6.6 microM, as shown by isothermal titration calorimetry. PhhR activates transcription from both the PP2827 and PP2078 promoters regardless of the absence or presence of aromatic amino acids, whereas PhhR stimulates transcription from certain positively regulated promoters (P(phhA), P(PP3122), P(PP3434), and P(hmg)) only in the presence of phenylalanine and tyrosine or their corresponding keto acids (i.e., phenylpyruvate and p-hydroxyphenylpyruvate). A surprising feature of PhhR-mediated transcriptional activation is that PhhR may bind to one or two upstream target sequences that are located at different distances from the RNA polymerase binding site. This allows PhhR to function as a class I regulator (target sites at -66/-83), a class II regulator (target sites around -40), as well as an enhancer protein (target sites >-128). When functioning as an enhancer protein, PhhR-mediated transcription is modulated by the integration host factor protein. PhhR represses transcription from its own promoter and the promoter of the paaY gene by steric hindrance.
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PMID:PhhR binds to target sequences at different distances with respect to RNA polymerase in order to activate transcription. 1978 50

The phhAB operon encodes a phenylalanine hydroxylase involved in the conversion of L-phenylalanine into L-tyrosine in Pseudomonas putida. The phhAB promoter is transcribed by RNA polymerase sigma-70 and is unusual in that the specific regulator PhhR acts as an enhancer protein that binds to two distant upstream sites (-75 to -92 and -132 to -149). There is an integration host factor (IHF) binding site that overlaps the proximal PhhR box, and, consequently, IHF acts as an inhibitor of transcription. Use of L-phenylalanine is compromised in a crp-deficient background due to reduced expression from the phhAB promoter. Electrophoretic mobility shift assays and DNase I footprinting assays reveal that Crp binds at a site centered at -109 only in the presence of cyclic AMP (cAMP). We show, using circular permutation analysis, that the simultaneous binding of Crp/cAMP and PhhR bends DNA to bring positive regulators and RNA polymerase into close proximity. This nucleoprotein complex promotes transcription from phhA only in response to L-phenylalanine.
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PMID:Involvement of the global Crp regulator in cyclic AMP-dependent utilization of aromatic amino acids by Pseudomonas putida. 2208 86