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)

Comparative studies of the effects of Mg2+ vs Na+ and of acetate (OAc-) vs Cl- on the kinetics of formation and dissociation of E. coli RNA polymerase (E sigma 70)-lambda PR promoter open complexes have been used to probe the mechanism of this interaction. Composite second-order association rate constants ka and first-order dissociation rate constants kd, and their power dependences on salt concentration SKa (SKa identical to d log ka/d log [salt]) and Skd (Skd identical to d log kd/d log [salt]), were determined in MgCl2 and NaOAc to compare with the results of Roe and Record (1985) in NaCl. Replacement of NaCl by MgCl2 reduces the magnitude of Ska 2-fold (Ska = -11.9 +/- 1.1 in NaCl; Ska = -5.2 +/- 0.3 in MgCl2) and (by extrapolation) drastically reduces the magnitude of ka at any specified salt concentration (e.g., approximately 10(6)-fold at 0.2 M). Replacement of NaCl by NaOAc does not significantly affect Ska (Ska = -12.0 +/- 0.7 in NaOAc) and (by extrapolation) increased ka by approximately 80-fold at any fixed [Na+]. In the absence of Mg2+, replacement of NaCl by NaOAc is found to increase the half-life of the open complex by approximately 560-fold at fixed [Na+] without affecting Skd [Skd = 7.6 +/- 0.1 in NaOAc; in NaCl, Skd = 7.7 +/- 0.2 (Roe & Record, 1985)]. Replacement of NaCl by MgCl2 drastically reduces both Skd and the half-life of the open complex at any salt concentration below approximately 0.2 M. Strikingly, Skd = 0.4 +/- 0.1 in MgCl2, indicating that the net uptake of Mg2+ ions in the kinetically significant steps in dissociation of the open complex is much smaller than that expected by analogy with the uptake of approximately 8 Na+ ions in the corresponding steps in NaCl. In NaCl/MgCl2 mixtures, at a constant [NaCl] in the range 0.1-0.2 M, initial addition of MgCl2 (0.5 mM less than or equal to [MgCl2] less than or equal to 1 mM) increases the half-life of the open complex; further addition of MgCl2 causes the half-life to decrease, though the effect of [MgCl2] on kd is always less than that predicted by a simple competitive model. The observed effects of MgCl2 on Skd and kd differ profoundly from those expected from the behavior of kd and Skd in NaCl and NaOAc and indicate that the role of Mg2+ in dissociation is not merely that of a nonspecific divalent competitor with RNAP for interactions with DNA phosphates and of a DNA helix-stabilizer, both of which should cause kd to increase monotonically with increasing [Mg2+].(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Roles of Mg2+ in the mechanism of formation and dissociation of open complexes between Escherichia coli RNA polymerase and the lambda PR promoter: kinetic evidence for a second open complex requiring Mg2+. 138 21

We describe procedures for preparing extracts of Xenopus oocytes, eggs, and somatic cells that will accurately transcribe class II genes. A variety of viral and Xenopus promoters direct the accurate initiation of transcription by RNA polymerase II in these extracts. Optimal ionic conditions (100-200 mM KCl, 12 mM MgCl2), template concentration (20-40 micrograms/ml), incubation time (30-60 min), and temperature (25 degrees C) for class II gene transcription are described.
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PMID:In vitro transcription by RNA polymerase II in extracts of Xenopus oocytes, eggs, and somatic cells. 141 31

Chloroplast tRNA(Glu) is a bifunctional molecule involved in both the early steps of chlorophyll synthesis and chloroplast protein biosynthesis. Recently the enzymes involved in these processes have been characterized from the green alga Chlamydomonas reinhardtii. In order to investigate whether transcription of the gene for the tRNA(Glu) cofactor would be a possible point of regulation for the biosynthesis of chlorophyll, a homologous in vitro transcription system for C. reinhardtii chloroplast RNA polymerase was developed. The enzymatic activity was partially purified by ion-exchange chromatography to separate it from nuclear RNA polymerases. The highest rate of synthesis was found at pH 7.9, 40 mM KCl, 9 mM MgCl2 and with 25 micrograms plasmid DNA containing the chloroplast tRNA gene per milliliter. The activity was not sensitive to high amounts of alpha-amanitin (500 micrograms/ml) and rifampicin, but was clearly inhibited by heparin. This system was used to undertake a promoter analysis of one of the two identical tRNA(Glu) gene copies found in the C. reinhardtii chloroplast genome (trnE1). The analyzed tRNA gene behaved like a single transcription unit driven by its own promoter. The transcript terminated in a run of four consecutive T residues downstream of the gene. The nucleotide sequence in the 5' region of the gene revealed several potential promoter elements with homology to known chloroplast promoters of the "-10 and -35 region" and the "Euglena promoter" types. Surprisingly, deletion of the complete 5' region did not affect in vitro transcription, while partial deletions of the 5' and 3' coding region totally abolished transcription. This indicates the presence of an internal control region previously found for genes transcribed by nuclear RNA polymerase III. Protein binding studies with the coding region of trnE1 using gel retardation assays demonstrated the formation of two differently sized complexes. In vitro transcription of the tRNA(Glu) gene in extracts prepared from light and dark grown algae failed to demonstrate any significant influence of light on the transcription reaction.
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PMID:Expression of the Chlamydomonas reinhardtii chloroplast tRNA(Glu) gene in a homologous in vitro transcription system is independent of upstream promoter elements. 141 80

We report on the discovery and isolation of DNA- and RNA-containing macromolecular nuclear complexes whose purified major DNA possessed electrophoretic mobilities of approximately 90 and approximately 25 kbp. The deoxyribonucleoprotein-ribonucleoprotein complexes contain RNA and DNA polymerase and primase activities and were isolated from nuclei of murine RAW117 large-cell lymphoma cells by restriction digestion with Msp-I, gentle extraction with solutions containing MgCl2, but without chelating agents, and low ionic strength gel electrophoresis. Two-dimensional (isoelectric focusing/M(r)) gel electrophoresis and silver staining of the proteins of the complexes after treatment with DNase I indicated the presence of approximately 30 protein components. In vitro DNA and RNA polymerase/primase assays showed that the DNP/RNP complexes had very high enzyme specific activities. Using the DNP/RNP complexes a discrete DNA polymerase alpha product of approximately 85 kbp was synthesized that was not synthesized in the presence of the DNA polymerase alpha inhibitor aphidicolin. RNA polymerase assays in the presence of excess alpha-amanitin indicated that the complexes possessed significant RNA polymerase I activity. Preparing the complexes at various times after the release of cells from a double thymidine block showed the complexes as well as the complex-associated enzyme activities to be cell-cycle dependent. The DNA and RNA polymerase-related activities were highest in late S phase, 7 and 9 h, respectively, after release from the double thymidine block. The complexes synthesized a specific in vitro DNA polymerase product using endogenous substrate and nucleotide precursors.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Nucleoprotein complexes released from lymphoma nuclei that contain the abl oncogene and RNA and DNA polymerase and RNA primase activities. 142 73

Escherichia coli RNA polymerase has two subsites, i and i + 1, for the binding of the first two substrates, and the first phosphodiester bond is formed between them during the initiation of transcription. Various studies have shown earlier that the inhibitor rifampicin has little effect, if any, on the formation of this phosphodiester bond. On an earlier occasion, we measured the distance of the i nucleotide from the rifampicin binding site on RNA polymerase using Forster's energy-transfer mechanism [Kumar & Chatterji (1990) Biochemistry 29,317]. In this paper, the 1-aminonaphthalene-5-sulfonic acid (AmNS) derivative of UTP in the presence of 10 mM MgCl2 was used as an energy donor, and its distance from rifampicin was estimated. The modified nucleotide (gamma-AmNS)-UTP binds to RNA polymerase with a Kd of 3 microM and has one binding site in the presence of Mg(II) ion. Fluorescence titration studies performed with or without an initiator indicated that (gamma-AmNS)-UTP exclusively binds to RNA polymerase at the (i + 1) site in the presence of Mg(II). Rifampicin was found to form a 1:1 complex with RNA polymerase bound to labeled UTP. Rifampicin and (gamma-AmNS)-UTP have a substantial spectral overlap with an energy-transfer efficiency close to 50%. Labeled UTP shows a decrease in its excited-state lifetime when bound to the enzyme; the transfer efficiency calculated from lifetime measurements was found to be lower than that estimated from steady-state spectral analysis. Time-resolved emission spectral analysis was carried out to differentiate between the free and bound UTP over the enzyme surface.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Proximity relationship between the active site of Escherichia coli RNA polymerase and rifampicin binding domain: a resonance energy-transfer study. 151 Sep 38

We constructed a 66 base-pair DNA template capable of supporting transcription by T7 RNA polymerase. This template had a psoralen cross-link downstream from a T7 promoter such that a 36 (+1) nucleotide transcript was synthesized at the time the T7 polymerase came to a stop at the cross-link. The stability of elongation complexes formed on this template, and the effect of different factors that are known to affect polymerase-DNA interactions was investigated by non-denaturing gel electrophoresis and gel filtration chromatography. We found that an elongation complex could lose its RNA component but the T7 polymerase still remained attached to the DNA template for extended periods of time (at least up to 18 h). This type of an elongation complex, bereft of its nascent RNA transcript, is called a quasi-elongation complex. DNase I footprinting within gel slices indicated that the polymerase molecules were arrested at the psoralen cross-link on the DNA template in the quasi-elongation complexes. The quasi-elongation complexes were found to be extremely stable in 0.5 M-NaCl and in 0.2 M-NaCl plus 60 mM-MgCl2, and could withstand temperatures up to 42 degrees C. The quasi-elongation complexes were destabilized by heparin and excess calf thymus DNA. Excess tRNA caused only a minimal degree of disruption. Non-promoter-containing plasmid DNAs did not have a destabilizing effect on the quasi-elongation complexes. Interestingly, it was observed that in a T7 ternary transcriptional complex arrested by a psoralen cross-link, the nascent RNA transcript could be stabilized from release by the presence in trans of a plasmid DNA bearing a T7 promoter and a T7 terminator. Such a stabilization against RNA release was not observed with plasmid DNAs containing either only a promoter or a terminator. The elongation complexes were stable during gel filtration through Sephacryl S-300 HR. However, it was found that 30% to 45% of the labeled RNA was retained during gel filtration as RNA that was apparently free from ternary complexes.
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PMID:Studies on the interaction of T7 RNA polymerase with a DNA template containing a site-specifically placed psoralen cross-link. II. Stability and some properties of elongation complexes. 194 45

Human brain hexokinase (hexokinase I) was produced in Escherichia coli from a synthetic gene under control of the bacteriophage T7 promoter. The expressed coding region derives from a human cDNA clone thought to specify hexokinase I based on amino acid sequence identity between the predicted translation product and hexokinase I from rat brain. The open reading frame from this cDNA was fused to the promoter and 5' flanking region of T7 gene 10, and expressed in E. coli by induction of T7 RNA polymerase. Induced cells contained a hexokinase activity and an abundant protein of apparent molecular weight 100,000, neither of which was present in cells lacking T7 RNA polymerase. Enzyme purified to near homogeneity consisted of a 100,000 Da protein, the size predicted from the nucleotide sequence of the expressed cDNA. The purified enzyme had Michaelis constants of 32 microM and 0.3 mM for glucose and ATP, respectively, and bound to rat liver mitochondria in the presence of MgCl2. Enzymatic activity was inhibited by glucose-6-P and this inhibition was relieved by inorganic phosphate. Deinhibition by phosphate is a property specific to brain hexokinase.
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PMID:Expression of human brain hexokinase in Escherichia coli: purification and characterization of the expressed enzyme. 204 17

The DNA-dependent RNA polymerase was purified from Rickettsia prowazekii, an obligate intracellular bacterial parasite. Because of limitation of available rickettsiae, the classical methods for isolation of the enzyme from other procaryotes were modified to purify RNA polymerase from small quantities of cells (25 mg of protein). The subunit composition of the rickettsial RNA polymerase was typical of a eubacterial RNA polymerase. R. prowazekii had beta' (148,000 daltons), beta (142,000 daltons), sigma (85,000 daltons), and alpha (34,500 daltons) subunits as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The appropriate subunits of the rickettsial RNA polymerase bound to polyclonal antisera against Escherichia coli core polymerase and E. coli sigma 70 subunit in Western blots (immunoblots). The enzyme activity was dependent on all four ribonucleoside triphosphates, Mg2+, and a DNA template. Optimal activity occurred in the presence of 10 mM MgCl2 and 50 mM NaCl. Interestingly, in striking contrast to E. coli, approximately 74% of the rickettsial RNA polymerase activity was associated with the rickettsial cell membrane at a low salt concentration (50 mM NaCl) and dissociated from the membrane at a high salt concentration (600 mM NaCl).
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PMID:Purification and partial characterization of the DNA-dependent RNA polymerase from Rickettsia prowazekii. 212 Jan 86

To identify the DNA sequences required for multidrug resistance (MDR1) gene transcription, we have optimized conditions for transcription of the MDR1 proximal promoter in vitro. Using HeLa cell nuclear extracts, the direction, initiation, and RNA polymerase II dependence of transcription in vitro accurately reflect events in cells. The DNA template concentration, reaction temperature, and MgCl2 concentration were critical parameters of the in vitro system. Using conditions optimized for these parameters, the effect of deletions in the 5' flanking region and deletions in sequences downstream of the initiation site were examined. We found that deletion of sequences 5' and 3' of the transcription initiation site modulated the level of transcription. Of particular interest was the deletion of exon 1 sequences +5 to +127, which completely inhibited accurately initiated MDR1 transcription. Reconstitution of the +5 site, used for initiation in vivo and in vitro, did not reverse the inhibition. MDR1 transcription was specifically inhibited by an oligonucleotide corresponding to sequences +46 to +58. Our data indicate that sequences both upstream and downstream of the transcription initiation site modulate the efficiency of the MDR1 proximal promoter.
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PMID:The human multidrug resistance gene: sequences upstream and downstream of the initiation site influence transcription. 228 76

Reverse transcriptase was purified from human immunodeficiency virus (HIV). It utilized the artificial primer-template poly(rA)-oligo(dT)12-18 more efficiently than activated calf thymus DNA, poly(rI)-oligo(dC)12-18, poly(rC)-oligo(dG)12-18, or poly(rCm)-oligo(dG)12-18. Maximum activity was observed at pH 7.0 to 7.6 in the presence of 5 mM MgCl2 and 100 mM KCl. 3'-Azido-3'-deoxythymidine triphosphate competed with dTTP for binding to HIV reverse transcriptase. Different kinetic constants were obtained with different primer-templates. Km and Ki values of 2.8 and 0.04 microM, respectively, were obtained with poly(rA)-oligo(dT)12-18. The corresponding values were 1.2 and 0.3 microM, respectively, with activated calf thymus DNA and 0.3 and 0.01 microM, respectively, with extracted virus and native template. Inhibition of the host cell DNA polymerases alpha and beta was considerably weaker. The Km and Ki values obtained with activated calf thymus DNA as the primer-template were 2.4 and 230 microM, respectively, for DNA polymerase alpha and 6.0 and 73 microM, respectively, for DNA polymerase beta. 3'-Azido-3'-deoxythymidine triphosphate could also serve as an alternate substrate for HIV reverse transcriptase. The resulting incorporation of 3'-azido-3'-deoxythymidine triphosphate into poly(rA)-oligo(dT)12-18 caused chain termination and premature deceleration of the reaction. The terminated primer could not be elongated when incubated with dTTP and HIV reverse transcriptase.
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PMID:3'-Azido-3'-deoxythymidine triphosphate as an inhibitor and substrate of purified human immunodeficiency virus reverse transcriptase. 244 66


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