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)

To begin to characterize biochemically the transcriptional activation systems in photosynthetic bacteria, the Rhodobacter capsulatus RNA polymerase (RNAP) that contains the sigma70 factor (R. capsulatus RNAP/sigma70) was purified and characterized using two classical sigma70 type promoters, the bacteriophage T7A1 and the RNA I promoters. Transcription from these promoters was sensitive to rifampicin, RNase, and monoclonal antibody 2G10 (directed against the Escherichia coli sigma70 subunit). Specific transcripts were detected in vitro for R. capsulatus cytochrome c2 (cycA) and fructose-inducible (fruB) promoters and genes induced in photosynthesis (puf and puc) and bacteriochlorophyll biosynthesis (bchC). Alignment of these natural promoters activated by R. capsulatus RNAP/sigma70 indicated a preference for the sequence TTGAC at the -35 region for strong in vitro transcription. To test the -35 recognition pattern, the R. capsulatus nifA1 promoter, which exhibits only three of the five consensus nucleotides at the -35 region, was mutated to four and five of the consensus nucleotides. Although the nifA1 wild type promoter showed no transcription, the double mutated promoter exhibited high levels of in vitro transcription by the purified R. capsulatus RNAP/sigma70 enzyme. Similarities and differences between the RNAPs and the promoters of R. capsulatus and E. coli are discussed.
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PMID:Characterization of the Rhodobacter capsulatus housekeeping RNA polymerase. In vitro transcription of photosynthesis and other genes. 934 Nov 73

These experiments sought to identify what form of RNA polymerase transcribes the P1 promoter for the Rhodobacter sphaeroides cytochrome c2 gene (cycA). In vitro, cycA P1 was recognized by an RNA polymerase holoenzyme fraction that transcribes several well-characterized Escherichia coli heat shock (sigma32) promoters. The in vivo effects of mutations flanking the transcription initiation site (+1) also suggested that cycA P1 was recognized by an RNA polymerase similar to E. coli Esigma32. Function of cycA P1 was not altered by mutations more than 35 bp upstream of position +1 or by alterations downstream of -7. A point mutation at position -34 that is towards the E. coli Esigma32 -35 consensus sequence (G34T) increased cycA P1 activity approximately 20-fold, while several mutations that reduced or abolished promoter function changed highly conserved bases in presumed -10 or -35 elements. In addition, cycA P1 function was retained in mutant promoters with a spacer region as short as 14 nucleotides. When either wild-type or G34T promoters were incubated with reconstituted RNA polymerase holoenzymes, cycA P1 transcription was observed only with samples containing either a 37-kDa subunit that is a member of the heat shock sigma factor family (Esigma37) or a 38-kDa subunit that also allows core RNA polymerase to recognize E. coli heat shock promoters (Esigma38). (R. K. Karls, J. Brooks, P. Rossmeissl, J. Luedke, and T. J. Donohue, J. Bacteriol. 180:10-19, 1998).
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PMID:Transcription of the Rhodobacter sphaeroides cycA P1 promoter by alternate RNA polymerase holoenzymes. 942 85

The Rhodobacter sphaeroides photosynthesis response regulator, PrrA, positively regulates cycA P2 expression. Deletion analysis has identified sequences within 73 bp upstream of the transcription initiation site that are required for the activation of cycA P2 by PrrA. A mutant form of the Rhodobacter capsulatus PrrA homologue, whose activity is independent of phosphorylation (RegA*), protects an approximately 26 bp region of cycA P2 that is centred at approximately -50 from DNase digestion, and activates transcription of a mutant -14T promoter with increased activity when using either R. sphaeroides RNA polymerase or Escherichia coli Esigma70. A 4 bp target site mutation that eliminated DNA binding and transcription activation by RegA* in vitro also abolished PrrA activation of cycA P2 transcription in vivo, indicating that this region contains a PrrA binding site. By analysing the behaviour of the -14T mutant cycA P2 promoter in vivo, we also found that PrrA uses the same target site to activate expression in both the presence and the absence of O2. However, the extent of transcription activation by PrrA at cycA P2 in vivo is greater under anaerobic conditions.
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PMID:Activation of the cycA P2 promoter for the Rhodobacter sphaeroides cytochrome c2 gene by the photosynthesis response regulator. 1056 21

Rhodobacter sphaeroides rpoE encodes a 19.2 kDa protein, sigma(E), related to members of the extra-cytoplasmic function subfamily of eubacterial RNA polymerase sigma factors. We demonstrate that sigma(E) directs transcription from rpoE P1, the promoter for the rpoEchrR operon, and from cycA P3, a promoter for the cytochrome c2 structural gene. Comparison of these sigma(E)-dependent promoters reveals significant sequence conservation in their -35 and -10 regions; however, rpoE P1 is over 80-fold stronger than cycA P3. Both promoters contain identical -35 hexamers, (-36)TGATCC(-31), that appear to constitute the preferred sequence, since any single base mutation in this region of cycA P3 reduces promoter function. The higher activity of rpoE P1 appears to reflect a better -10 region, (-13)TAAGA(-9), as it contains four out of five of the nucleotides found to be important to sigma(E)-dependent transcription. We also propose that ChrR acts as an inhibitor of sigma(E), since these two proteins can form a complex, and DeltachrR mutations increase sigma(E)-dependent transcription. ChrR is believed to respond to a signal from tetrapyrrole biosynthesis because loss of function mutations in chrR lead to cohemin resistance. Based on our observations, we present a model in which cohemin resistance is conferred by increasing sigma(E) activity.
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PMID:The Rhodobacter sphaeroides ECF sigma factor, sigma(E), and the target promoters cycA P3 and rpoE P1. 1061 Jul 60

The Rhodobacter sphaeroides extra cytoplasmic function sigma factor, sigma(E), directs transcription of promoters for the cycA gene (cycA P3) and the rpoEchrR operon (rpoE P1). These genes encode the periplasmic electron carrier cytochrome c(2) and sigma(E)/ChrR, respectively. Using in vitro transcription assays with purified R. sphaeroides core RNA polymerase and sigma(E), we show that ChrR is sufficient to inhibit sigma(E)-dependent transcription. Inhibition is proposed to proceed through a binding interaction, since sigma(E) and ChrR form a 1:1 complex that can be purified when expressed at high levels in Escherichia coli. Active preparations of ChrR and the sigma(E)/ChrR complex each contain stoichiometric zinc. Removal of zinc from ChrR or a single amino acid substitution that abolishes zinc binding, results in a protein that is incapable of inhibiting sigma(E) activity or forming a complex with the sigma factor, indicating that metal binding is important to ChrR activity. Treatment of ChrR with the thiol-modifying reagent p-hydroxymecuriphenylsulfonic acid results in the release of about one mole of zinc per mole of protein. Furthermore, two N-terminal cysteine residues are protected from reaction with the thiol-specific reagent dithionitrobenzoic acid until zinc is removed, suggesting that these residues may be involved in zinc binding. These data indicate that ChrR is a specific anti-sigma factor of sigma(E) that requires zinc for function. Based on amino acid sequence similarity, we propose that ChrR is part of a family of similar anti-sigma factors that are found in alpha and gamma proteobacteria.
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PMID:The importance of zinc-binding to the function of Rhodobacter sphaeroides ChrR as an anti-sigma factor. 1167 34

We have identified a second RpoH homolog, RpoH(II), in the alpha-proteobacterium Rhodobacter sphaeroides. Primary amino acid sequence comparisons demonstrate that R. sphaeroides RpoH(II) belongs to a phylogenetically distinct group with RpoH orthologs from alpha-proteobacteria that contain two rpoH genes. Like its previously identified paralog, RpoH(I), RpoH(II) is able to complement the temperature-sensitive phenotype of an Escherichia coli sigma(32) (rpoH) mutant. In addition, we show that recombinant RpoH(I) and RpoH(II) each transcribe two E. coli sigma(32)-dependent promoters (rpoD P(HS) and dnaK P1) when reconstituted with E. coli core RNA polymerase. We observed differences, however, in the ability of each sigma factor to recognize six R. sphaeroides promoters (cycA P1, groESL(1), rpoD P(HS), dnaK P1, hslO, and ecfE), all of which resemble the E. coli sigma(32) promoter consensus. While RpoH(I) reconstituted with R. sphaeroides core RNA polymerase transcribed all six promoters, RpoH(II) produced detectable transcripts from only four promoters (cycA P1, groESL(1), hslO, and ecfE). These results, in combination with previous work demonstrating that an RpoH(I) mutant mounts a typical heat shock response, suggest that while RpoH(I) and RpoH(II) have redundant roles in response to heat, they may also have roles in response to other environmental stresses.
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PMID:Activity of Rhodobacter sphaeroides RpoHII, a second member of the heat shock sigma factor family. 1688 39

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