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)

Studies on bacterial RNA polymerases have divided the initiation pathway into three steps, namely (i) promoter binding to form the closed complex; (ii) DNA melting to form an open complex, and (iii) messenger RNA initiation. Potassium permanganate was used to detect DNA melting by mammalian RNA polymerase II in vitro. Closed complexes formed in a rate-limiting step that was stimulated by the activator GAL4-VP16. Adenosine triphosphate was then hydrolyzed to rapidly melt the DNA within the closed complex to form an open complex. Addition of nucleoside triphosphates resulted in the melted bubble moving away from the start site, completing initiation.
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PMID:Polymerase II promoter activation: closed complex formation and ATP-driven start site opening. 131 Mar 61

Footprinting studies with the purine-modifying reagent dimethyl sulfate and with the single-stranded DNA probing reagent potassium permanganate were carried out in isolated mitochondria from rat liver. Dimethyl sulfate footprinting allowed the detection of protein-DNA interactions within the rat analogues of the human binding sites for the transcription termination factor mTERF and for the transcription activating factor mt-TFA. Although mTERF contacts were localized only at the boundary between the 16S rRNA/tRNA(Leu)UUR genes, multiple mtTFA contacts were detected. Contact sites were located in the light and the heavy strand promoters and, in agreement with in vitro footprinting data on human mitochondria, between the conserved sequence blocks (CSB) 1 and 2 and inside CSB-1. Potassium permanganate footprinting allowed detection of a 25-base pair region entirely contained in CSB-1 in which both strands were permanganate-reactive. No permanganate reactivity was associated with the other regions of the D-loop, including CSB-2 and -3, and with the mTERF contact site. We hypothesize that the single-stranded DNA at CSB-1 may be due to a profound helix distortion induced by mtTFA binding or be associated with a RNA polymerase pause site. In any case the location in CSB-1 of the 3' end of the most abundant replication primer and of the 5' end of the prominent D-loop DNA suggests that protein-induced DNA conformational changes play an important role in directing the transition from transcription to replication in mammalian mitochondria.
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PMID:Identification by in Organello footprinting of protein contact sites and of single-stranded DNA sequences in the regulatory region of rat mitochondrial DNA. Protein binding sites and single-stranded DNA regions in isolated rat liver mitochondria. 755 32

An RNA polymerase II activator often contains several regions that contribute to its potency, an organization ostensibly analogous to the modular architecture of promoters and enhancers. The regulatory significance of this parallel organization has not been systematically explored. We considered this problem by examining the activation domain of the Epstein-Barr virus transactivator ZEBRA. We performed our experiments in vitro so that the activator concentrations, stabilities, and affinities for DNA could be monitored. ZEBRA and various amino-terminal deletion derivatives, expressed in and purified from Escherichia coli, were assayed in a HeLa cell nuclear extract for the ability to activate model reporter templates bearing one, three, five, and seven upstream ZEBRA binding sites. Our data show that ZEBRA contains four modules that contribute to its potency in vitro. The modules operate interchangeably with promoter sites to determine the transcriptional response such that the loss of modules can be compensated for by increasing promoter sites. Potassium permanganate footprinting was used to show that transcriptional stimulation is a consequence of the activator's ability to promote preinitiation complex assembly. Kinetic measurements of transcription complex assembly in a reconstituted system indicate that ZEBRA promotes formation of a subcomplex requiring the TFIIA and TFIID fractions, where TFIIA acts as an antirepressor. We propose a model in which the concentration of DNA-bound activation modules in the vicinity of the gene initiates synergistic transcription complex assembly.
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PMID:The ZEBRA activation domain: modular organization and mechanism of action. 841 94

Potassium permanganate (KMnO4) footprinting in the absence and presence of magnesium (Mg2+) at the lambda PR promoter identified two different open complexes with Escherichia coli E sigma 70 RNA polymerase (designated RPo1 and RPo2). The single-stranded region in RPo1 (formed in the absence of Mg2+) was at most 12 bases long, whereas that in RPo2 (formed in the presence of Mg2+) spanned at least 14 bases. Only in RPo2 did the single-stranded region extend to the start point of transcription (+1, +2). These results provide a structural basis for the requirement for uptake of Mg2+ in the formation of RPo2 from RPo1, as deduced from kinetic studies at this promoter.
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PMID:Two open complexes and a requirement for Mg2+ to open the lambda PR transcription start site. 842 2

Potassium permanganate oxidation of pyrimidine bases is often used to probe single-stranded regions in functional DNA-protein complexes. However, so far reactivity of these bases in double-stranded DNA has not been studied quantitatively. We have investigated the kinetics of oxidation of pyrimidines in supercoiled pDS3 plasmid dsDNA by quantitative KMnO4 footprinting, in connection with parallel studies on the effect of Mg2+ on kinetics of oxidation of individual thymines in the single-stranded region of the open transcription complex of Escherichia coli RNA polymerase at a cognate Pa promoter contained in this plasmid. Rate constants of oxidation for pyrimidines, kj, in selected regions of pDS3 DNA, including Pa promoter, were determined under single-hit reaction conditions in the absence and presence of 10 mM MgCl2. Their values appeared to be sequence-dependent and were: (i) the largest for Ts in 5'TA3' and 5'TC3' steps, while 2-4 times smaller for 5'-adjacent ones in TT(A,G,C) and TTT(A) runs, (ii) for Cs in 5'TC3' steps 2-4 fold smaller than for adjacent Ts, and (iii) in the presence of Mg2+ generally larger by a sequence-dependent factor: in 5'TC3' steps of about 2 and 4 for Ts and Cs, respectively, in 5'TA3' steps of TTA and TTTA sequences for 3'-terminal Ts of about 3, while for their 5'-neighbors o tinctly smaller value of about 2. Comparison of kj data for corresponding Ts located between +1 and -10 regions of Pa promoter in dsDNA and in ssDNA form in the open transcription complex, reported elsewhere, demonstrates that reactivity of pyrimidines in dsDNA is by 2-3 orders of magnitude smaller. The effect of Mg2+ in dsDNA is interpreted in terms of electrostatic barrier to diffusion of MnO4- on DNA surface, which is lowered by diffusive binding of these ions to backbone phosphates, involving also sequence-specific contacts with bases in the minor and major grooves of B-DNA.
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PMID:Effect of Mg2+ on kinetics of oxidation of pyrimidines in duplex DNA by potassium permanganate. 1173 20

Phage T4 early promoters are transcribed in vivo and in vitro by the Escherichia coli RNA polymerase holoenzyme Esigma(70). We studied in vitro the effects of the T4 anti-sigma(70) factor AsiA on the activity of several T4 early promoters. In single-round transcription, promoters motB, denV, mrh.2, motA wild type and UP element-deleted motA are strongly resistant to inhibition by AsiA. The alpha-C-terminal domain of Esigma(70) is crucial to this resistance. DNase I footprinting of Esigma(70) and Esigma(70)AsiA on motA and mrh.2 shows extended contacts between the holoenzyme with or without AsiA and upstream regions of these promoters. A TG --> TC mutation of the extended -10 motif in the motA UP element-deleted promoter strongly increases susceptibility to inhibition by AsiA, but has no effect on the motA wild-type promoter: either the UP element or the extended -10 site confers resistance to AsiA. Potassium permanganate reactivity shows that the two structure elements are not equivalent: with AsiA, the motA UP element-deleted promoter opens more slowly whereas the motA TC promoter opens like the wild type. Changes in UV laser photoreactivity at position +4 on variants of motA reveal an analogous distinction in the roles of the extended -10 and UP promoter elements.
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PMID:Phage T4 early promoters are resistant to inhibition by the anti-sigma factor AsiA. 1513 Jan 21

Potassium permanganate (KMnO4) has widely been used in genomic footprinting assays to map unusual gene structures, including the melting DNA block in transcriptional elongation that results from promoter-proximal pausing of RNA polymerase (Pol) II complexes. Although it has been assumed that DNA-bound proteins do not protect underlying nucleic acids from KMnO4 modifications, we provide evidence herein that this chemical can readily be used to detect nuclear factor loading at a promoter when using optimized conditions. Moreover, by comparing parallel KMnO4 and dimethylsulfate (DMS) in vivo footprintings, we show that the utilization of KMnO4 in combination with another chemical probe maximizes the detection of factor occupancy at a DNA regulatory region, thus providing a better opportunity to define the actual profiles of DNA-protein contacts at given genomic sites in living cells.
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PMID:Potassium permanganate as a probe to map DNA-protein interactions in vivo. 1516 30

A-factor (2-isocapryloyl-3R-hydroxymethyl-gamma-butyrolactone) triggers streptomycin production by inducing the transcription of strR, encoding the pathway-specific transcriptional activator, through signal transduction in the A-factor regulatory cascade in Streptomyces griseus. AdpA, one of the key transcriptional activators in the cascade, bound two upstream activation sites, approximately at nucleotide positions -270 and -50 with respect to the transcriptional start point of strR, as determined by gel mobility shift assays and DNase I footprinting. Transcriptional analysis of the strR promoter with mutated AdpA-binding sites showed that both sites were required for full transcriptional activation of strR by AdpA. Potassium permanganate footprinting showed that AdpA assisted RNA polymerase in forming an open complex at an appropriate position for transcriptional initiation of strR. Nine transcriptional units within the streptomycin biosynthesis gene cluster, including the strR-aphD operon, depended on StrR, indicating that StrR is the pathway-specific transcriptional activator for the whole gene cluster. Consistent with this, expression of strR under the control of a constitutively expressed promoter in an adpA null mutant caused the host to produce streptomycin.
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PMID:Transcriptional control by A-factor of strR, the pathway-specific transcriptional activator for streptomycin biosynthesis in Streptomyces griseus. 1607 4

Escherichia coli sigma70-dependent promoters have typically been characterized as either -10/-35 promoters, which have good matches to both the canonical -10 and the -35 sequences or as extended -10 promoters (TGn/-10 promoters), which have the TGn motif and an excellent match to the -10 consensus sequence. We report here an investigation of a promoter, P(minor), that has a nearly perfect match to the -35 sequence and has the TGn motif. However, P(minor) contains an extremely poor sigma70 -10 element. We demonstrate that P(minor) is active both in vivo and in vitro and that mutations in either the -35 or the TGn motif eliminate its activity. Mutation of the TGn motif can be compensated for by mutations that make the -10 element more canonical, thus converting the -35/TGn promoter to a -35/-10 promoter. Potassium permanganate footprinting on the nontemplate and template strands indicates that when polymerase is in a stable (open) complex with P(minor), the DNA is single stranded from positions -11 to +4. We also demonstrate that transcription from P(minor) incorporates nontemplated ribonucleoside triphosphates at the 5' end of the P(minor) transcript, which results in an anomalous assignment for the start site when primer extension analysis is used. P(minor) represents one of the few -35/TGn promoters that have been characterized and serves as a model for investigating functional differences between these promoters and the better-characterized -10/-35 and extended -10 promoters used by E. coli RNA polymerase.
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PMID:Escherichia coli RNA polymerase recognition of a sigma70-dependent promoter requiring a -35 DNA element and an extended -10 TGn motif. 1701 80

We previously reported that the P1 promoter of topA encoding topoisomerase I of Escherichia coli is activated in response to oxidative stress, in a Fis-dependent manner. Here we show that Fis regulation of topA varies with the intracellular concentrations of Fis. Thus, when Fis levels are low, hydrogen peroxide treatment results in topA activation, whereas at high Fis levels hydrogen peroxide treatment renders topA P1 inactive. In vivo DMS footprinting indicates that only at low Fis levels, when exposed to the stress, the region of the topA promoter changes and P1 becomes active. Potassium permanganate experiments indicate that low levels of Fis activate P1 transcription by facilitating the formation of open complexes, while high levels of this protein shut off the promoter. DNase I footprinting show that Fis binds the promoter region of topA at eight sites with different affinities. One low affinity site overlaps the -10, -35 hexamers of RNA polymerase. We propose that in response to oxidative stress, when present at low levels, Fis binds the promoter region of topA at its high affinity sites, thereby facilitating the recruitment of RNA polymerase to P1, while at high levels, Fis occupies the low affinity sites as well, and thus prevents the binding of RNA polymerase. Our results indicate that the oxidative stress response varies in response to changes in growth phase and nutritional environment.
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PMID:Differential regulation of Escherichia coli topoisomerase I by Fis. 1723 26


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