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)

Positive control of the wild-type Klebsiella pneumoniae nifH promoter by the NifA protein requires that NifA is bound at the upstream activator sequence (UAS). By introducing base substitutions at -15 to -17 in the RNA polymerase recognition sequence of the nifH promoter, positive control by a form of NifA unable to bind to the UAS was greatly increased when compared to the wild-type promoter. Transcriptional activation still required the rpoN encoded sigma factor and was initiated at the same nucleotide as in the wild-type promoter. Mutations at -15 to -17 suppressed the requirement that the UAS should be located on the correct face of the DNA helix with respect to the RNA polymerase recognition sequence in order that titration of NifA and efficient activation occur. This result supports the suggestion that upstream bound NifA interacts with the RNA polymerase-RpoN complex. To examine the minimal carboxy terminal sequences required for the positive control function of NifA a series of carboxy terminal deletions were constructed. Efficient positive control at a UAS-independent promoter was only observed in deletions which did not extend beyond the proposed boundary separating the carboxy terminal NifA DNA-binding domain from its central domain.
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PMID:Mutations in the RNA polymerase recognition sequence of the Klebsiella pneumoniae nifH promoter permitting transcriptional activation in the absence of NifA binding to upstream activator sequences. 254 10

Hybrid 5' regulatory regions were constructed in which the upstream activator sequence (UAS) and promoter of various nif genes were exchanged with the upstream regulatory sequence (URS) of the fdhF gene from Escherichia coli. They were analysed for their regulatory response under different growth conditions with the aid of fdhF'-'lacZ or nif'-'lacZ fusions. Placement of the UAS from the Bradyrhizobium japonicum nifH gene in front of the spacer (DNA region between URS and promoter) plus promoter from fdhF renders fdhF expression activatable by the Klebsiella pneumoniae NIFA protein, both under aerobic and anaerobic conditions. This excludes the possibility that the spacer of the fdhF5' flanking region contains a site recognized by a putative oxygen- or nitrate-responsive repressor. There was also considerable activation by NIFA of fdhF expression in a construct lacking the nifH UAS but containing the fdhF spacer plus promoter. Further experimental evidence suggests that this reflects a direct interaction between NIFA and RNA polymerase at the ntrA-dependent promoter. A second set of hybrid constructs in which the URS from fdhF (E. coli) was placed in front of the nifD spacer plus promoter from B. japonicum or in front of the K. pneumoniae nifH, nifU, nifB spacers and promoters, delivered inactive constructs in the case of the nifD, nifU and nifB genes. However, a nifH'-'lacZ fusion preceded by its own spacer and promoter plus the foreign fdhF URS displayed all the regulatory characteristics of fdhF expression, i.e. anaerobic induction with formate and repression by oxygen and nitrate. Although it is not known why only one out of the four nif promoters could be activated by the fdhF URS, this result nevertheless demonstrates that the various regulatory stimuli affecting expression of fdhF in E. coli have their target at the upstream regulatory sequence.
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PMID:Construction of chimaeric promoter regions by exchange of the upstream regulatory sequences from fdhF and nif genes. 266 22

Activation of transcription at the Klebsiella pneumoniae nifLA promoter requires the phosphorylated form of the positive control protein NTRC, together with RNA polymerase modified by the alternative sigma factor sigma 54. Dimethylsulphate and potassium permanganate were used as probes to analyse the interaction of NTRC and sigma 54-RNA polymerase with supercoiled nifLA promoter DNA in vitro. In contrast to the glnAp2 promoter, sigma 54 holoenzyme did not protect guanine residues in the nifLA promoter from methylation in the absence of the activator. We propose that NTRC stabilizes the interaction of sigma 54-RNA polymerase with the -24, -12 region, in addition to its role in catalysing open complex formation. Phosphorylated NTRC binds to two sites located greater than 100 nucleotides upstream of the -24, -12 region; it also induces hyper-methylation of a G residue at -23. Enhanced methylation at -23 is not co-operative with the binding of activator to the upstream sites and may account for the ability of NTRC, when present at high concentration, to activate transcription in the absence of the upstream binding sites. The insertion of spacer mutations at -86 indicates that transcriptional activation of the nifLA promoter at low NTRC concentrations is face-of-the-helix dependent, both in vivo and in vitro. We propose that correct positioning of activator molecules at the upstream binding sites stabilizes the interaction of sigma 54-RNA polymerase with the downstream region via the formation of a DNA loop.
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PMID:Transcriptional activation of the Klebsiella pneumoniae nifLA promoter by NTRC is face-of-the-helix dependent and the activator stabilizes the interaction of sigma 54-RNA polymerase with the promoter. 268 43

Transcription from the Klebsiella pneumoniae and Rhizobium meliloti nifH promoters requires the positive control protein NifA and the alternative sigma factor sigma 54, encoded by the rpoN gene. Transcription from the K. pneumoniae nifH promoter is fully dependent upon NifA bound at the upstream activator sequence (UAS) whereas the R. meliloti nifH promoter can be efficiently activated in the absence of this sequence and can also be activated by a mutant form of NifA unable to bind the UAS. The in vivo interaction of RNA polymerase-sigma 54 with these promoters was examined using dimethyl sulphate footprinting. The R. meliloti nifH promoter but not the K. pneumoniae nifH promoter showed sigma 54-dependent methylation protection of guanine residues at -14, -25 and -26, the most conserved nucleotides characteristic of sigma 54-dependent promoters. A mutant derivative of the K. pneumoniae nifH promoter bearing transitions at positions from -15 to -17 showed sigma 54-dependent methylation protection of guanines -13, -24 and -25. The enhanced interaction of the RNA polymerase-sigma 54 with this mutant promoter correlates with its increased level of activation by a form of NifA unable to bind the UAS. Use of in vivo KMnO4 footprinting to detect single-stranded pyrimidine residues and in vivo methylation protection demonstrated that the sigma 54-dependent protection observed in the R. meliloti and mutant K. pneumoniae nifH promoter results from the formation of a closed promoter complex. The isomerization of the pre-existing closed complex to an open promoter form, as judged by the local denaturation of promoter DNA which rendered sequences from +5 to -10 reactive towards KMnO4, was shown to be fully dependent on NifA. We propose a model in which the fidelity of activation of sigma 54-dependent promoters relies on a weak activator-independent interaction of RNA polymerase-sigma 54 with the promoter. A specific interaction of the appropriate activator with its respective UAS is then required for the positive control protein to facilitate open complex formation.
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PMID:In vivo studies on the interaction of RNA polymerase-sigma 54 with the Klebsiella pneumoniae and Rhizobium meliloti nifH promoters. The role of NifA in the formation of an open promoter complex. 268 31

The nucleotide sequence of the Klebsiella pneumoniae ntrA gene has been determined. NtrA encodes a 53,926 Dalton acidic polypeptide; a calculated molecular weight which is significantly lower than that determined by SDS polyacrylamide gel analysis. NtrA is followed by another open-reading frame (orf) of at least 75 amino acids. In the spacer region between ntrA and orf there are no apparent transcription termination or promoter sequences and therefore orf may be co-transcribed with ntrA. Previous authors have proposed that NtrA could act as an RNA polymerase sigma factor but the NtrA amino acid sequence does not show a high level of homology to any known sigma factor. However analysis of sequences of five sigma factors from E. coli and B. subtilis has identified two conserved sequences at the C-terminal end of all these polypeptides. These sequences resemble those found in known site-specific DNA-binding domains and may be involved in recognition of conserved -35 and -10 promoter sequences. A similar pair of sequences is present at the C-terminus of NtrA and could play a role in recognition of ntr-activatable promoters.
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PMID:The nucleotide sequence of the nitrogen-regulation gene ntrA of Klebsiella pneumoniae and comparison with conserved features in bacterial RNA polymerase sigma factors. 299

Initiation of DNA replication from the Escherichia coli origin, oriC, is dependent on an RNA polymerase-mediated transcription event. The function of this RNA synthetic event in initiation, however, remains obscure. Since control of the synthesis of this RNA could serve a key role in the overall initiation process, transcription regulatory sites within and near oriC were identified using the galK fusion vector system. Our results confirm the existence of a transcription termination signal within oriC, first identified by Hansen et al. (1981), for the 16 kd transcript that is transcribed counterclockwise towards oriC. Termination is shown to be 92% efficient. A similar approach led to the detection of transcription termination within the chromosomal replication origin of Klebsiella pneumoniae. Approximately 50% of the E. coli 16 kd transcripts appear to terminate before reaching oriC between the XhoI (+416 bp) and the HindIII (+243 bp) sites. The predominant 3' ends of RNA that enter oriC, as determined by SI nuclease mapping, were located at positions +20 +/- 2, +23 +/- 2, +37, +39, +52, +66, +92, and +107. These termination sites, which map cl to RNA . DNA junctions identified by Kohara et al. (1985), appear as triplets and quadruplets. The E. coli oriC Pori-L promoter described in in vitro transcription studies by Lother and Messer (1981) was not detected in this study in either wildtype cells or isogenic dnaA mutants at the nonpermissive temperature. A new promoter activity, Pori-R1, was identified within the E. coli origin in the clockwise direction.
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PMID:Transcription termination within the Escherichia coli origin of DNA replication, oriC. 301 76

We have determined the nucleotide sequence of the xylR gene for a transcriptional activator for the degradative pathway of aromatic hydrocarbons on the TOL plasmid from Pseudomonas putida. The 1698-bp sequence for a 566-amino acid (aa) protein (Mr 63741) was identified as the XylR-encoding sequence. Three regions in XylR show homology to Klebsiella pneumoniae NtrC and NifA, both of which are transcriptional activators for the ntr and nif genes involved in the nitrogen metabolism. The central region of XylR (aa 234-473) corresponds to the region that was proposed to interact with RNA polymerase having a sigma factor, NtrA [Drummond et al., EMBO J. 5 (1986) 441-447]. The C-terminal region (aa 515-558) has a putative DNA-binding structure. A short segment proximal to the central region (aa 211-229) is thought to be an interdomain linker. No amino acid homology was found in the N-terminal regions among these proteins. These findings suggest the interaction of XylR with an NtrA in the transcriptional activation of the degradative pathway.
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PMID:Nucleotide sequence of the regulatory gene xylR of the TOL plasmid from Pseudomonas putida. 316 74

Mutants of Klebsiella aerogenes able to express the hutUH operon in the absence of positive effectors were isolated and characterized. These mutations improve the hutUH promoter (PUH) by changing the -10 region to match the consensus sequence more closely. These mutations also affect another, oppositely oriented promoter in this region, PC. Although the mutations lie far outside PC, they cause PC to be inactive, apparently because binding of RNA polymerase to the PUH promoter blocks the overlapping PC site. Thus, in the mutants, RNA polymerase bound at the strong (mutant) PUH site effectively repress the PC promoter.
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PMID:RNA polymerase as a repressor of transcription in the hut(P) region of mutant Klebsiella aerogenes histidine utilization operons. 317 Apr 91

The Pseudomonas putida TOL plasmid pWWO carries an operon that specifies a meta-cleavage pathway for the catabolism of benzoate and toluates whose transcription is positively regulated by the xylS gene product. Stimulation of transcription of the operon is thought to result from activation of this protein by pathway substrates/effectors. In the present study, overexpression of the xylS gene has led to identification of the regulator as a 33 kDa protein. Overexpression of xylS also resulted in partially constitutive, i.e. effector-independent expression of the meta-cleavage operon. Determination of the polynucleotide sequence of the xylS gene revealed amino acid sequence homology with several DNA binding proteins, particularly with the araC products of Escherichia coli and Salmonella typhimurium and with the nifA and ntrC products of Klebsiella pneumoniae. Homologous sequences were mainly located in an alpha-helix-turn-alpha-helix domain of the polypeptide. Interestingly, amino acid sequence homology was also found with sigma factors of E. coli (ntrA and htpR products) and Bacillus subtilis (spoIIG and phage SPOI Gp34 products) and other RNA polymerase core-interacting proteins, such as the E. coli nusA product.
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PMID:The xylS gene positive regulator of TOL plasmid pWWO: identification, sequence analysis and overproduction leading to constitutive expression of meta cleavage operon. 347 26

The nucleotide sequence of the Azotobacter vinelandii ntrA gene has been determined. It encodes a 56916 Dalton acidic polypeptide (AvNtrA) with substantial homology to NtrA from Klebsiella pneumoniae (KpNtrA) and Rhizobium meliloti (RmNtrA). NtrA has been shown to act as a novel RNA polymerase sigma factor but the predicted sequence of AvNtrA substantiates our previous analysis of KpNtrA in showing no substantial homology to other known sigma factors. Alignment of the predicted amino acid sequences of AvNtrA, KpNtrA and RmNtrA identified three regions; two showing greater than 50% homology and an intervening sequence of less than 10% homology. The predicted protein contains a short sequence near the centre with homology to a conserved region in other sigma factors. The C-terminal region contains a region of homology to the beta' subunit of RNA polymerase (RpoC) and two highly conserved regions one of which is significantly homologous to known DNA-binding motifs. In A. vinelandii, ntrA is followed by another open reading frame (ORF) which is highly homologous to a comparable ORF downstream of ntrA in K. pneumoniae and R. meliloti.
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PMID:The nucleotide sequence of the sigma factor gene ntrA (rpoN) of Azotobacter vinelandii: analysis of conserved sequences in NtrA proteins. 348 23


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