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)

While the components of the initiation complex at an RNA polymerase II basal promoter have been well characterized, few mechanistic studies have focused on how upstream DNA-binding, transcriptional activators influence protein assembly at the initiation site. Our analysis of basal transcription on both the simian virus 40 and adenovirus major late promoters demonstrates that two slow steps in initiation of transcription are the assembly of the general transcription factors TFIID and TFIIB onto the template DNA. On the simian virus 40 major late promoter, the rate of initiation complex formation is dramatically increased in the presence of the cellular transcriptional activator LSF. Direct analysis by band mobility shift assays demonstrates that LSF has no effect on the rate of binding, or the stability of TFIID on the promoter, predicting that LSF would not affect the template commitment step. Rather, kinetic analyses demonstrate that LSF reduces the lag in the rate of initiation complex formation attributable to the slow addition of TFIIB and suggest that LSF increases the rate of association of TFIIB with the committed template. In addition, LSF increases the total number of transcription complexes in long term assays, which is also consistent with LSF increasing the rate of association of TFIIB, where TFIIB is not saturating. These results indicate a mechanism for the activation of the initiation of RNA polymerase II transcription by one upstream activating protein, LSF. This mechanism may also be applicable to other activators that function in cases where limiting concentrations of TFIIB in the cell dictate slow binding of TFIIB.
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PMID:Activation of RNA polymerase II transcription by the specific DNA-binding protein LSF. Increased rate of binding of the basal promoter factor TFIIB. 131 10

We constructed mercury resistance operon-luciferase (mer-lux) transcriptional fusion plasmids to evaluate in vivo gene expression rates of the mer structural gene promoter (PTPCAD) of transposon Tn21. In vivo gene expression kinetics corresponded well with those previously determined in vitro, yielding an apparent K0.5 for Hg(II)-stimulated induction by MerR of 9.3 x 10(-8) M with the same ultrasensitive threshold effect seen in vitro. We also used the mer-lux fusions to elucidate subtle variations in promoter activity brought about by altered superhelicity. Binding of inducer [Hg(II)] to the transcriptional activator MerR is known to result in DNA distortion and transcriptional activation of the mer operon; it has recently been demonstrated that this distortion is a consequence of MerR-Hg(II)-induced local DNA unwinding to facilitate RNA polymerase open complex formation at PTPCAD. Since negative supercoiling results in DNA unwinding similar to this MerR activation, we hypothesized that a global increase in plasmid supercoiling would facilitate MerR-mediated activation and compromise MerR-mediated repression, while removal of plasmid supercoils would compromise MerR's ability to induce transcription and facilitate its ability to repress transcription. Indeed, we found that increased negative supercoiling results in increased gene expression rates and decreased supercoiling results in reduced gene expression rates for the induced, repressed, and derepressed conditions of PTPCAD. Thus, luciferase transcriptional fusions can detect subtle variations in initial rates of gene expression in a real-time, nondestructive assay.
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PMID:A mer-lux transcriptional fusion for real-time examination of in vivo gene expression kinetics and promoter response to altered superhelicity. 133 70

Transcription of Rhodobacter capsulatus genes encoding the nitrogenase polypeptides (nifHDK) is repressed by fixed nitrogen and oxygen. R. capsulatus nifA1 and nifA2 encode identical NIFA proteins that activate transcription of nifHDK and other nif genes. In this study, we report that nifA1-lacZ and nifA2-lacZ fusions are repressed in the presence of NH3 and activated to similar levels under nitrogen-deficient conditions. This nitrogen-controlled activation was dependent on R. capsulatus ntrC (which encodes a transcriptional activator) but not rpoN (which encodes an RNA polymerase sigma factor). We have used primer extension analyses of nifA1, nifA2 and nifH and deletion analyses of nifA1 and nifA2 upstream regions to define likely promoters and cis upstream activation sequences required for nitrogen control of these genes. Primer extension mapping confirmed that ntrC but not rpoN is required for nifA1 and nifA2 activation, and that nifA1 and nifA2 do not possess typical RPON-activated promoters.
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PMID:Analysis of the promoters and upstream sequences of nifA1 and nifA2 in Rhodobacter capsulatus; activation requires ntrC but not rpoN. 137 28

Escherichia coli cells harbouring the rpoA341 mutation produce an RNA polymerase which transcribes inefficiently certain operons subject to positive control. Here, we demonstrate that the rpoA341 allele also prevents lysogenization of the host strain by bacteriophage lambda, a process dependent upon the action of two phage-encoded activators. This phenomenon was shown to arise from an inability to establish an integrated prophage rather than a failure to maintain the lysogenic state. The inability of the rpoA341 host to support lysogenization could be completely reversed by CII-independent expression of int and cI in trans. These results led us to propose that the inhibition of lysogenization arises from a defective interaction between the phage lambda transcriptional activator CII and the mutant RNA polymerase at the phage promoters pI and pE. Finally, we also provide genetic evidence for impaired transcription of the cI gene from the CI-activated promoter, pM in the rpoA341 background.
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PMID:Involvement of the Escherichia coli RNA polymerase alpha subunit in transcriptional activation by the bacteriophage lambda CI and CII proteins. 145 17

We have previously shown that transcription of the Xenopus U6 snRNA gene by RNA polymerase III is stimulated in injected Xenopus oocytes by an activator element termed the DSE, which contains an octamer sequence. Data presented here reveal that the DSE contains, in addition, a GC-rich sequence capable of binding Sp1. Both elements are required to obtain wild-type levels of U6 transcription in vivo. The Xenopus U6 DSE exhibits optimal activation properties only when positioned at its normal location upstream from the start site. The U6 Sp1 motif binds the mammalian Sp1 transcriptional activator independently of the Oct-1 protein in vitro. Those mutations that lead to a reduced transcription level in vivo abolish the binding of Sp1 in vitro. Thus, transcriptional stimulation through the Xenopus U6 Sp1 motif is likely to be mediated by a protein with DNA-binding specificity identical to mammalian Sp1. These findings support the notion that RNA polymerase II and III transcription complexes share transactivators.
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PMID:A factor with Sp1 DNA-binding specificity stimulates Xenopus U6 snRNA in vivo transcription by RNA polymerase III. 145 50

Many baculovirus early genes and insect genes transcribed by RNA polymerase II have a conserved transcription start site sequence (CAGT) located downstream of a consensus TATA box. To examine the functions and interactions of these two motifs in initiating accurately positioned basal transcription, a 43-nt synthetic promoter was synthesized from the TATA box and start site sequences of the gp64 early promoter from the Orgyia pseudotsugata multicapsid nuclear polyhedrosis virus (OpMNPV). The synthetic promoter initiated accurately and was also transactivated by the baculovirus transcriptional activator, IE1. To determine the roles of sequences within the 43-nt synthetic promoter, a series of linker-scanning and spacing mutations were analyzed for transcriptional activity, start site selection, and transactivation. Linker-scanning mutations were examined in vivo by transient expression and reporter gene assays. To examine transcription start site selection, promoter constructs were used for in vitro transcription in nuclear extracts from uninfected Spodoptera frugiperda (Sf9) cells. In vivo and in vitro analyses show that the TATA box, and not the start site CAGT, is the primary element controlling start site selection. Substitution of the conserved start site CAGT sequence resulted in a reduction of both reporter gene activity and in vitro transcripts, although transcripts initiated accurately. Data from linker-scanning and spacing mutations indicate that the conserved start site CAGT sequences are not required for accurate initiation but sequences at the start site play an important role in initiation efficiency.
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PMID:A synthetic early promoter from a baculovirus: roles of the TATA box and conserved start site CAGT sequence in basal levels of transcription. 151 59

The herpes simplex virus type 1 (HSV-1) ICP4 protein is a transcriptional activator of many eucaryotic RNA polymerase II promoters. The HSV-1 thymidine kinase gene (tk) promoter is induced by ICP4 and contains binding sites for the cellular transcription factors TFIID, Sp1, and CCAAT-binding proteins, each of which affects expression of the tk gene. In this study, the effects of mutations in these sites on the transcription of tk in the presence and absence of ICP4 were determined during viral infection. Only the TATA box was necessary for efficient expression in the presence of ICP4; however, ICP4 apparently can still induce tk transcription even when the TATA box is disrupted. Alteration of the Sp1 sites had a minor effect on ICP4-induced expression in comparison to a large effect in the absence of ICP4, indicating that ICP4 can operationally substitute for the function of the transcription factor Sp1. In addition, tk was still expressed with the kinetics of an early gene in the absence of binding sites for Sp1 and CCAAT-binding proteins.
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PMID:Herpes simplex virus transactivator ICP4 operationally substitutes for the cellular transcription factor Sp1 for efficient expression of the viral thymidine kinase gene. 184 84

Myxobacterial hemagglutinin (MBHA) is a major developmentally induced protein that accumulates during the period of cellular aggregation of the fruiting bacterium Myxococcus xanthus. In this study, DNA sequences mediating the transcriptional regulation of mbhA have been identified. Examination of nucleotide sequences upstream of the start site for mbhA transcription has indicated a region of DNA that bears strong homology to the consensus sequence for promoters recognized by the sigma 54 holoenzyme form of RNA polymerase of Escherichia coli and other eubacteria. Deletion of this sequence completely abolished mbhA transcription. Additionally, a cis-acting DNA element, affecting the efficiency of mbhA transcription, has been mapped within a region of DNA 89 to 276 nucleotides upstream of the sigma 54-like sequence. Transposon insertions, mapping within the cis element, drastically reduced mbhA transcriptional activity. These observations suggest that transcription of mbhA requires a productive interaction between a form of RNA polymerase that recognizes a sigma 54-like sequence and a transcriptional activator that binds to DNA sequences upstream of the mbhA promoter.
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PMID:Transcription of the myxobacterial hemagglutinin gene is mediated by a sigma 54-like promoter and a cis-acting upstream regulatory region of DNA. 185 Apr 3

Expression of Bacillus subtilis phage phi 29 late genes requires the transcriptional activator protein p4. This activator binds to a region of the late A3 promoter spanning nucleotides -56 to -102 relative to the transcription start site, generating a strong bending Tin the DNA. In this work the target sequences recognized by protein p4 in the phage phi 29 late A3 promoter have been characterized. The binding of protein p4 to derivatives of the late A3 promoter harbouring deletions in the protein p4 binding site has been studied. When protein p4 recognition sequences were altered, the activator could only bind to the promoter in the presence of RNA polymerase. This strong cooperativity in the binding of protein p4 and RNA polymerase to the promoter suggests the presence of direct protein-protein contacts between them.
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PMID:Identification of the sequences recognized by phage phi 29 transcriptional activator: possible interaction between the activator and the RNA polymerase. 190 53

RNA polymerase II lacking the fourth and seventh largest subunits (pol II delta 4/7) was purified from Saccharomyces cerevisiae strain rpb-4, in which the gene for the fourth largest subunit is deleted. pol II delta 4/7 was indistinguishable from wild-type pol II (holoenzyme) in promoter-independent initiation/chain elongation activity (400-800 nmol of nucleotide incorporated/10 min/mg of protein at 22 degrees C), in rate of chain elongation (20-25 nucleotides/s), and in the recognition of pause sites in the DNA template. In contrast to pol II holoenzyme, pol II delta 4/7 was inactive in promoter-directed initiation of transcription in vitro. The addition of an equimolar complex of the fourth and seventh largest subunits, purified from pol II holoenzyme by ion-exchange chromatography in the presence of urea, restored promoter-directed initiation activity to pol II delta 4/7. The transcriptional activator protein Gal4-VP16 could also elicit promoter-directed initiation by pol II delta 4/7 from a promoter with a Gal4 binding site. Complementation was observed between extracts of strain rpb-4, lacking the fourth largest subunit, and strain Y260-1, with a defect in the largest subunit. These extracts were individually inactive, but a mixture would support promoter-directed initiation. The fourth and seventh largest subunits may, therefore, shuttle between polymerase molecules.
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PMID:Two dissociable subunits of yeast RNA polymerase II stimulate the initiation of transcription at a promoter in vitro. 198 24


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