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 promoter region preceding the hutUH operon in Klebsiella aerogenes contains two oppositely oriented, overlapping promoters. In the absence of catabolite gene activator protein-cyclic AMP (CAP-cAMP), transcription proceeds primarily from the backward-oriented promoter (Pc), whose function has not yet been determined, and only very weakly from the forward hutUH promoter, hutUp. In the presence of CAP-cAMP, Pc is repressed and transcription from hutUp is favored. Two protein components required for this in vitro transcription system, RNA polymerase (RNAP) and CAP, were purified from K. aerogenes and were shown to be functionally interchangeable with the corresponding proteins from Escherichia coli, suggesting that E. coli RNAP could be used to study some aspects of hut transcription. We showed that a gradual activation of hutUp (by increasing concentrations of CAP, cAMP, or glycerol) resulted in a parallel repression of Pc, arguing in favor of a direct competition between the two promoters. The presence of a DNA sequence resembling the consensus for CAP-binding sites and centered at nucleotide -82 (relative to hutUp) initially suggested that a primary role of CAP was to repress Pc, thereby indirectly activating hutUp. However, the relatively slow formation of open complexes at Pc, even in the absence of CAP-cAMP, showed that Pc is a weak promoter and likely to be a poor competitor for RNAP. The observed dominance of Pc over hutUp suggested that the latter is an even weaker promoter. Thus, repression of Pc would not be sufficient to cause the observed increase in hutUp activity, and the CAP-cAMP complex must play a direct role in the activation of hutUp.
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PMID:In vitro transcription of the histidine utilization (hutUH) operon from Klebsiella aerogenes. 184 33

Recognition of -24/-12-type promoters by RNA polymerase requires a special sigma factor, sigma 54 (RpoN NtrA GlnF). In the nitrogen-fixing soybean symbiont Bradyrhizobium japonicum, two functional, highly conserved rpoN genes (rpoN1 and rpoN2) were identified and sequenced. The two predicted B. japonicum RpoN protein sequences were 87% identical, and both showed different levels of homology to the RpoN proteins of other bacteria. Downstream of rpoN2 (but not of rpoN1), two additional open reading frames were identified that corresponded to open reading frames located at similar positions in Klebsiella pneumoniae and Pseudomonas putida. Both B. japonicum rpoN genes complemented the succinate- and nitrate-negative phenotypes of a Rhizobium meliloti rpoN mutant. B. japonicum strains carrying single or double rpoN mutations were still able to utilize C4-dicarboxylates as a carbon source and histidine, proline, or arginine as a nitrogen source, whereas the ability to assimilate nitrate required expression of at least one of the two rpN genes. In symbiosis both rpoN genes could replace each other functionally. The rpoN1/2 double mutant induced about twice as many nodules on soybeans as did the wild type, and these nodules lacked nitrogen fixation activity completely. Transcription of a nifH'-'lacZ fusion was not activated in the rpoN1/2 mutant background, whereas expression of a fixR'-'lacZ fusion in this mutant was affected only marginally. By using rpoN'-'lacZ fusions, rpoN1 expression was shown to be activated at least sevenfold in microaerobiosis as compared with that in aerobiosis, and this type of regulation involved fixLJ. Expression of rpoN2 was observed under all conditions tested and was increased fivefold in an rpoN2 mutant. The data suggested that the rpoN1 gene was regulated in response to oxygen, whereas the rpoN2 gene was negatively autoregulated.
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PMID:Bradyrhizobium japonicum has two differentially regulated, functional homologs of the sigma 54 gene (rpoN). 199 12

A mutation, serine 170 to alanine, in the proposed ATP binding site of the activator protein NTRC prevents transcriptional activation at sigma 54-dependent promoters both in vivo and in vitro. The rate of phosphorylation of the mutant protein by NTRB and the stability of mutant NTRC-phosphate were similar to those of wild-type NTRC. The phosphorylated mutant protein shows only a slight decrease in affinity (around 2-fold) for tandem NTRC binding sites in the Klebsiella pneumoniae nifL promoter suggesting that the mutation primarily influences the positive control function of NTRC. Moreover the mutant protein is trans dominant to the wild-type protein with respect to transcriptional activation at both the glnAp2 and nifL promoters. In vitro footprinting experiments reveal that the mutant protein is unable to catalyse isomerisation of closed promoter complexes between sigma 54-RNA polymerase and the nifL promoter to open promoter complexes. However, the mutant protein retains the ability to increase the occupancy of the -24, -12 region by sigma 54-RNA polymerase, forming closed complexes at the nifL promoter, which are not detectable in the absence of NTRC. These data support a model in which the activator influences the formation of closed complexes at the nifL promoter in addition to its role in catalysing open complex formation.
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PMID:Influence of a mutation in the putative nucleotide binding site of the nitrogen regulatory protein NTRC on its positive control function. 204 69

A new gene whose product is required for the production of formate hydrogenlyase (FHL) has been identified in Escherichia coli. This gene, termed fhlB, maps between the frdA (94.4 min) and argI (96.6 min) genes on the E. coli chromosome and is transcribed in a clockwise direction toward argI. Biochemical analysis of an FhlB- mutant, strain SE-2011 [phi(fhlB-lacZ+)], revealed that the mutant lacks formate dehydrogenase activity associated with FHL (FDH-H) and hydrogenase activity. As a result of these defects, fermentative hydrogen production and hydrogen uptake reactions were undetectable in strain SE-2011. Fumarate reductase activity of this mutant was also reduced to about 15% of the levels of the parent (strain MC4100), and strain SE-2011 did not produce succinate as a fermentation end product. Regulation of expression of the fhlB gene, studied as production of beta-galactosidase activity by strain SE-2011, revealed that the operon is expressed at low levels under aerobic conditions. Under anaerobic growth conditions, this activity increased by two- to threefold. Addition of formate enhanced the differential rate of synthesis of the fhlB gene product to as high as 130 U of beta-galactosidase specific activity per microgram of cell protein, but only under anaerobic conditions. Formate-dependent expression of phi(fhlB-lacZ+) required the sigma 54 subunit of RNA polymerase and the fhlA gene product. The concentration of formate required for maximum expression of the fhlB gene was about 15 mM; this value decreased to about 3 mM in the presence of plasmid pSE-133, which carries the fhlA gene in a multicopy plasmid. DNA sequence analysis of the fhlA gene showed that the FhlA protein is 686 amino acids long and has an anhydrous molecular weight of 78,086. On the basis of sequence homology with other transcriptional activators such as NtrC, HydG, and Klebsiella pneumoniae NifA proteins, the FhlA protein was deduced to be a transcriptional activator controlling the production of FHL. It is proposed that formate interacts with the FhlA protein and that this active complex initiates transcription of the fhlB gene. The FhlA and FhlB proteins act as a cascade in regulating the production of FDH-H and the FHL-linked hydrogenase and ultimately the production of FHL and fermentative hydrogen.
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PMID:Genetic regulation of formate hydrogenlyase of Escherichia coli: role of the fhlA gene product as a transcriptional activator for a new regulatory gene, fhlB. 211 3

The nucleotide (nt) sequence of the Rhizobium leguminosarum nifH promoter region contains a consensus promoter, a consensus upstream activator sequence (UAS), a pseudo (psi) promoter and a psi UAS. We mapped the transcription start point for the consensus promoter sequence by primer extension. This promoter differs from the consensus in one of the four supposedly invariant nt and can be activated by the Klebsiella pneumoniae nifA product in Escherichia coli. Under these conditions the psi promoter and psi UAS do not function. A low-copy-number plasmid construct containing the psi UAS as well as the consensus UAS delayed the onset of symbiotic nitrogen fixation in nodules induced on Pisum sativum. Studies of high-copy-number nifH promoter constructs showed that partial deletion of the consensus UAS does not alter the ability to inhibit nitrogen fixation by titration of NifA suggesting that NifA can also complex with RNA polymerase containing the alternative sigma-factor RpoN.
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PMID:The nifH promoter region of Rhizobium leguminosarum: nucleotide sequence and promoter elements controlling activation by NifA protein. 218 38

The Klebsiella pneumoniae nifU promoter is positively controlled by the NifA protein and requires a form of RNA polymerase holoenzyme containing the rpoN encoded sigma factor, sigma 54. Occupancy of the K. pneumoniae nifU promoter by NifA was examined using in vivo dimethyl sulphate footprinting. Three binding sites for NifA (Upstream Activator Sequences, UASs 1, 2 and 3) located at -125, -116 and -72 were identified which conform to the UAS consensus sequence TGT-N10-ACA. An additional NifA binding site was identified at position -90. The UASs located at -125 (UAS1) and -116 (UAS2) overlap and do not appear to bind NifA as independent sites. They may represent a NifA binding site interacting with two NifA dimers. UAS3 is located at -72, and abuts a binding site for integration host factor (IHF) and is not normally highly occupied by NifA. In the absence of IHF UAS3 showed increased occupancy by NifA. Mutational and footprinting analysis of the three UASs indicates (1) IHF and NifA can compete for binding and that this competition influences the level of expression from the nifU promoter (2) that UAS2 is a principle sequence of the UAS 1,2 region required for activation and (3) that none of the NifA binding sites interacts with NifA independently. In vivo KMnO4 footprinting demonstrated that NifA catalyses open complex formation at the nifU promoter. IHF was required for maximal expression from the nifU and nifH promoters in Escherichia coli, and for the establishment of a Nif+ phenotype in E. coli from the nif plasmid pRD1.
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PMID:Activation of the Klebsiella pneumoniae nifU promoter: identification of multiple and overlapping upstream NifA binding sites. 218 62

The regulatory protein NIFA activates transcription of nitrogen fixation (nif) operons by the sigma 54 holoenzyme form of RNA polymerase. NIFA from Klebsiella pneumoniae activates transcription from the nifH promoter in vitro; in addition, the integration host factor, IHF, binds between the nifH promoter and an upstream binding site for NIFA. We demonstrate here that IHF greatly stimulates NIFA-mediated activation of nifH transcription in vitro and thus that the two factors are functionally synergistic. Electron micrographs indicate that IHF bends the DNA in the nifH promoter regulatory region. Although IHF binds close to the nifH promoter, it does not directly stimulate binding of sigma 54 holoenzyme. Rather, the IHF-induced bend may facilitate productive contacts between NIFA and sigma 54 holoenzyme that lead to the formation of open complexes. IHF binds to nif promoter regulatory regions from a variety of organisms within the phylum "purple bacteria," suggesting a general ability to stimulate NIFA-mediated activation of nif transcription.
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PMID:The integration host factor stimulates interaction of RNA polymerase with NIFA, the transcriptional activator for nitrogen fixation operons. 220 75

We determined the nucleotide sequence of gene 1 of Klebsiella phage K11, which is a member of the T7 group of phages. The largest open reading frame corresponds to a polypeptide with 906 amino acids and a molecular weight of 100,383 daltons. The deduced amino acid sequence of this polypeptide shows 71% homology to the T7 RNA polymerase (the product of T7 gene 1), 72% homology to the T3 RNA polymerase and 27% homology to the SP6 RNA polymerase. Divergent evolution was clearly most pronounced in the amino-terminal portion.
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PMID:The gene for Klebsiella bacteriophage K11 RNA polymerase: sequence and comparison with the homologous genes of phages T7, T3, and SP6. 237 Aug 50

The protein nitrogen regulator I (NRI)-phosphate is known to activate the initiation of transcription of the Escherichia coli glnA gene. This activation is facilitated by the binding of the protein to NRI-specific sites located upstream of the sigma 54-dependent glnA promoter. To determine whether binding of NRI-phosphate to upstream sites is sufficient for activation, we placed several promoters not normally activated by NRI-phosphate downstream of NRI binding sites and measured activation in intact cells and in an in vitro transcription system. We found that the sigma 70-dependent lac promoter was not activated, that the sigma 54-dependent Klebsiella pneumoniae nifH promoter was weakly activated, and that a nifH promoter altered in the RNA polymerase binding site was almost as well activated as the glnA promoter. We conclude that the sensitivity of the susceptible promoter depends on the presence of NRI binding sites, but that the presence of bound NRI-phosphate upstream of a promoter is not sufficient for activation of transcription by RNA polymerase. This activation is determined by the structure of the RNA polymerase binding site. We suggest that sigma 54-but not sigma 70-dependent promoters are susceptible to activation by NRI-phosphate and that the nucleotide sequence of the sigma 54-RNA polymerase binding site is an important determinant of the efficiency of activation.
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PMID:Role of the promoter in activation of transcription by nitrogen regulator I phosphate in Escherichia coli. 240 58

We describe the cloning of an ntrC gene of Agrobacterium tumefaciens C58 by interspecific complementation of an Escherichia coli ntrC mutant. Restriction mapping and Southern blot analysis of the complementing clone identified a 1.7-kb EcoRI-PvuII DNA fragment whose sequence was determined. Analysis of this sequence revealed coding regions corresponding to a complete ntrC gene and the C-terminal region of an ntrB gene. Amino acid sequence comparisons of A. tumefaciens NTRC protein with NTRC sequences from Rhizobium meliloti, Bradyrhizobium sp. (Parasponia), Klebsiella pneumoniae, E. coli, and Salmonella typhimurium show strong sequence conservation supporting DNA hybridization data, demonstrating strong evolutionary homology among ntrC genes of Rhizobiaceae. The C58 NTRC protein has been identified, by 35S-labeling, in a T7 RNA polymerase (pT7-7) expression vector system.
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PMID:Identification, cloning, and sequence analysis of the nitrogen regulation gene ntrC of Agrobacterium tumefaciens C58. 252 Aug 24


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