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)

An open reading frame located upstream of the bacterioferritin gene in Escherichia coli encodes a hypothetical 64-residue protein [Andrews, S.C., Harrison, P.C., & Guest, J.R. (1989) J. Bacteriol. 171, 3940-3947)]. The spacing of the four cysteine residues in this hypothetical protein is identical to that in a region of NIFU, a [2Fe-2S] protein found in nitrogen-fixing bacteria [Fu, W., Jack, R.F., Morgan, T.V., Dean, D.R., & Johnson, M.K. (1994) Biochemistry 33, 13455-13463)]. The NIFU-like E. coli gene was cloned and overexpressed with a C-terminal "His tag" in E. coli using the T7 RNA polymerase/promoter system, and the protein was purified by metal-chelate affinity chromatography. UV-vis absorption and EPR spectra together with iron and amino acid analyses conclusively established that this NIFU-like E. coli protein contains one [2Fe-2S] cluster which can exist in at least two oxidation levels: +2 for the as-purified protein, and +1 for dithionite-reduced protein. Size-exclusion chromatography established that this His-tagged [2Fe-2S] protein is monomeric in solution. Affinity chromatography demonstrated specific complex formation between bacterioferritin (Bfr) and this NIFU-like [2Fe-2S] protein, which is dubbed Bfd. An open reading frame encoding a homologous Bfd is located near a Bfr gene in at least one other bacterium. Bfd may, therefore, constitute a general redox and/or regulatory component participating in the iron storage or mobilization functions of Bfr.
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PMID:A [2Fe-2S] protein encoded by an open reading frame upstream of the Escherichia coli bacterioferritin gene. 863 72

The PII protein has been considered pivotal to the dual cascade regulating ammonia assimilation through glutamine synthetase activity. Here we show that PII, encoded by the glnB gene, is not always essential; for instance upon ammonia deprivation of a glnB deletion strain, glutamine synthetase can be deadenylylated as effectively as in the wild-type strain. We describe a new operon, glnK amtB, which encodes a homologue of PII and a putative ammonia transporter. We cloned and overexpressed glnK and found that the expressed protein had almost the same molecular weight as PII, reacted with polyclonal PII antibody, and was 67% identical in terms of amino acid sequence with Escherichia coli PII. Like PII, purified GlnK can activate the adenylylation of glutamine synthetase in vitro, and, in vivo, the GlnK protein is uridylylated in a glnD-dependent fashion. Unlike PII, however, the expression of glnK depends on the presence of UTase, nitrogen regulator I (NRI), and absence of ammonia. Because of a NRI and a sigma N (sigma 54) RNA polymerase-binding consensus sequence upstream from the glnK gene, this suggests that glnK is regulated through the NRI/NRII two-component regulatory system. Indeed, in cells grown in the presence of ammonia, glutamine synthetase deadenylylation upon ammonia depletion depended on PII. Possible regulatory implications of this conditional redundancy of PII are discussed.
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PMID:An alternative PII protein in the regulation of glutamine synthetase in Escherichia coli. 884 40

The gene encoding the principal sigma factor from Synechococcus sp. strain PCC 7002 was isolated and characterized. The Synechococcus sp. strain PCC 7002 sigA gene encodes a protein of 375 amino acids (43 center dot 7 kDa) that is required for viability under normal growth conditions. The SigA protein was overproduced in Escherichia coli and the purified protein was used to raise polyclonal antiserum in rabbits. This antiserum was used in immunoblot analyses of partially purified RNA polymerase from Synechococcus sp. strain PR6000. The probable in vivo translational start site was identified by a comparison of amino acid sequencing results obtained with SigA proteins overproduced in E. coli with immunoblot analyses of SigA protein in crude preparations of RNA polymerase from the cyanobacterium. The sigA gene is encoded on a transcript of 1700 bases that initiates 496 nucleotides upstream from the probable in vivo translational start site. The abundance of sigA transcripts decreases rapidly after the removal of combined nitrogen from the growth medium.
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PMID:The sigA gene encoding the major sigma factor of RNA polymerase from the marine cyanobacterium Synechococcus sp. strain PCC 7002: cloning and characterization. 893 8

Nitrogen regulatory protein C (NtrC) contacts a bacterial RNA polymerase from distant enhancers by means of DNA loops and activates transcription by allowing polymerase to gain access to the template DNA strand. It was shown that NtrC from Salmonella typhimurium must build large oligomers to activate transcription. In contrast to eukaryotic enhancer-binding proteins, most of which must bind directly to DNA, some NtrC dimers were bound solely by protein-protein interactions. NtrC oligomers were visualized with scanning force microscopy. Evidence of their functional importance was provided by showing that some inactive non-DNA-binding and DNA-binding mutant forms of NtrC can cooperate to activate transcription.
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PMID:Unusual oligomerization required for activity of NtrC, a bacterial enhancer-binding protein. 907 26

The expression of genes transcribed by the RNA polymerase with the alternative sigma factor sigma 54 (E sigma 54) is absolutely dependent on activator proteins that bind to enhancer-like sites, located far upstream from the promoter. These unique prokaryotic proteins, known as enhancer-binding proteins (EBP), mediate open promoter complex formation in a reaction dependent on NTP hydrolysis. The best characterized proteins of this family of regulators are NtrC and NifA, which activate genes required for ammonia assimilation and nitrogen fixation, respectively. In a recent IRBM course (@ontiers of protein structure prediction," IRBM, Pomezia, Italy, 1995; see web site http://www.mrc-cpe.cam.uk/irbm-course95/), one of us (J.O.) participated in the elaboration of the proposal that the Central domain of the EBPs might adopt the classical mononucleotide-binding fold. This suggestion was based on the results of a new protein fold recognition algorithm (Map) and in the mapping of correlated mutations calculated for the sequence family on the same mononucleotide-binding fold topology. In this work, we present new data that support the previous conclusion. The results from a number of different secondary structure prediction programs suggest that the Central domain could adopt an alpha/beta topology. The fold recognition programs ProFIT 0.9, 3D PROFILE combined with secondary structure prediction, and 123D suggest a mononucleotide-binding fold topology for the Central domain amino acid sequence. Finally, and most importantly, three of five reported residue alterations that impair the Central domain. ATPase activity of the E sigma 54 activators are mapped to polypeptide regions that might be playing equivalent roles as those involved in nucleotide-binding in the mononucleotide-binding proteins. Furthermore, the known residue substitution that alter the function of the E sigma 54 activators, leaving intact the Central domain ATPase activity, are mapped on region proposed to play an equivalent role as the effector region of the GTPase superfamily.
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PMID:A proposed architecture for the central domain of the bacterial enhancer-binding proteins based on secondary structure prediction and fold recognition. 907 Apr 37

Little information is available on the effects of UVA (320-400 nm radiation) on transcription. We examined the effect of UVA on RNA synthesis in isolated chicken liver nuclei. Nuclei in air or nitrogen were irradiated with UVA, and the RNA synthesis induced by endogenous RNA polymerase was estimated under conditions in which little or no initiation occurs. Incorporation of [3H]UMP into the acid-insoluble fraction was used as the measure of RNA synthesis in the nuclei. In air the amount of synthesized RNA decreased with increasing UVA fluence. In contrast, in nitrogen UVA had little effect on RNA synthesis. Sodium azide and histidine, which effectively scavenge singlet oxygen (1O2) as well as hydroxyl radicals (.OH), protected the nuclei from inhibition of RNA synthesis; whereas, sodium formate and dimethyl sulfoxide, both of which much more effectively scavenge .OH than 1O2, had no protective effect. These findings provide a strong indication that 1O2 is involved in the inhibition of RNA synthesis. In addition, RNA polymerase II-dependent synthesis (in the nucleoplasm) was much more sensitive to UVA than RNA polymerase I-dependent synthesis (in the nucleolus).
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PMID:Effect of UVA on RNA synthesis in isolated chicken liver nuclei. 916 76

Scanning force microscopy (SFM) has been used to study transcriptional activation of Escherichia coli RNA polymerase x sigma 54 (RNAP x sigma 54) at the glnA promoter by the constitutive mutant NtrC(D54E,S160F) of the NtrC Protein (nitrogen regulatory protein C). DNA-protein complexes were deposited on mica and images were recorded in air. The DNA template was a 726 bp linear fragment with two NtrC binding sites located at the end and about 460 bp away from the RNAP x sigma 54 glnA promoter. By choosing appropriate conditions the structure of various intermediates in the transcription process could be visualized and analyzed: (1) different multimeric complexes of NtrC(D54E,S160F) dimers bound to the DNA template; (2) the closed complex of RNAP x sigma 54 at the glnA promoter; (3) association between DNA bound RNAP x sigma 54 and NtrC(D54E,S160F) with the intervening DNA looped out; and (4) the activated open promoter complex of RNAP x sigma 54. Measurements of the DNA bending angle of RNAP x sigma 54 closed promoter complexes yielded an apparent bending angle of 49(+/-24) degrees. Under conditions that allowed the formation of the open promoter complex, the distribution of bending angles displayed two peaks at 50(+/-24) degrees and 114(+/-18) degrees, suggesting that the transition from the RNAP x sigma 54 closed complex to the open complex is accompanied by an increase of the DNA bending angle.
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PMID:Transcriptional activation via DNA-looping: visualization of intermediates in the activation pathway of E. coli RNA polymerase x sigma 54 holoenzyme by scanning force microscopy. 923 16

The nitrogen fixation protein NifA is a member of the protein family activating transcription by the alternative eubacterial sigmaN (sigma54) RNA polymerase holoenzyme. Binding sites for NifA, upstream activator sequences (UASs), are remotely located. Interaction between holoenzyme bound in a closed promoter complex and NiFA is facilitated by bending of the intervening DNA by integration host factor (IHF). We have examined NifA contact with the Klebsiella pneumoniae nifH promoter UAS in the presence and absence of holoenzyme and IHF. Footprints with UV light were made on 5-BrdU-substituted DNA and DNase I and laser UV footprints on conventional DNA templates. Results establish that the consensus thymidine residues of the UAS motif 5'-TGT are in close proximity to NifA. Reactivity suggests that each UAS thymidine is not structurally equivalent. Titration of NifA binding to the UAS in the presence or absence of the closed promoter complex indicates that the interaction of NifA with the UAS is not strongly co-operative with holoenzyme or IHF, a result supportive of an activation mechanism not reliant upon simple recruitment of factors to the promoter. Laser footprints demonstrated that holoenzyme suppressed reactivity of promoter consensus -14, -15 and -16 T residues, indicating close contact. Binding of holoenzyme resulted in a specific increase in 5-BrdU reactivity at -9 within the holoenzyme binding site, likely reflecting DNA distortion. Enhanced -9 reactivity required sigmaNN-terminal sequences that are necessary for activation. Since T-9 is melted in open complexes the closed complex appears poised for melting. Open promoter complex formation was accompanied by a distinct change in laser footprint signal at -11, consistent with the view that nucleation of strand separation occurs within or close to the -12 promoter element.
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PMID:Nucleoprotein complex formation by the enhancer binding protein nifA. 925 7

We have examined the effects on transcription initiation of promoter and enhancer strength and of the curvature of the DNA separating these entities on wild-type and mutated enhancer-promoter regions at the Escherichia coli sigma54-dependent promoters glnAp2 and glnHp2 on supercoiled and linear DNA. Our results, together with previously reported observations by other investigators, show that the initiation of transcription on linear DNA requires a single intrinsic or induced bend in the DNA, as well as a promoter with high affinity for sigma54-RNA polymerase, but on supercoiled DNA requires either such a bend or a high affinity promoter but not both. The examination of the DNA sequence of all nif gene activator- or nitrogen regulator I-sigma54 promoters reveals that those lacking a binding site for the integration host factor have an intrinsic single bend in the DNA separating enhancer from promoter.
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PMID:DNA bending and the initiation of transcription at sigma54-dependent bacterial promoters. 927 63

Expression of urease, which is encoded by the ureABC operon, is regulated in response to nitrogen availability in Bacillus subtilis. Three ureABC promoters were identified in primer extension experiments and by examination of beta-galactosidase expression from ure-lacZ fusions. P1, a low-level constitutive promoter, lies immediately upstream of ureA. The P2 promoter is transcribed by the E sigmaH form of RNA polymerase and initiates transcription 270 bp upstream of the ureA start codon. The transcriptional start site for the sigmaA-dependent P3 promoter is located 839 bp upstream of the ureA start codon. To identify transcription factors that control ureABC expression, regulation of the P2 and P3 promoters was examined in wild-type and mutant strains. During rapid growth in minimal medium containing glucose and amino acids, CodY represses expression of the P2 and P3 promoters 30- and 60-fold, respectively. TnrA activates expression of the P3 promoter 10-fold in nitrogen-limited cells, while GlnR represses transcription from the P3 promoter 55-fold during growth on excess nitrogen. Expression of the ureABC operon increases 10-fold at the end of exponential growth in nutrient sporulation medium. This elevation in expression results from the relief of CodY-mediated repression during exponential growth and increased sigmaH-dependent transcription during stationary phase.
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PMID:Expression of the Bacillus subtilis ureABC operon is controlled by multiple regulatory factors including CodY, GlnR, TnrA, and Spo0H. 928 5


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