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)

The present paper describes a rapid, specific and sensitive method for quantitating ribonucleoside triphosphates (ATP and UTP) in cell extracts. The principle of the method is based on the synthesis of a ribonucleotide polymer in the presence of UTP, ATP and poly(dA-dT) as template. A method for calculation is also described, making the determination of UTP and ATP pool sizes in the cells possible under the same experimental conditions. The calculation takes into account the isotope dilution effect caused by the intracellular ATP. Our experiments show that the neutralized perchloric acid soluble fraction of human tonsillar lymphocytes contains no inhibitors for the RNA polymerase test. According to our results, this cell extract contains 80 pmol of UTP and 340 pmol of ATP per mug RNA.
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PMID:Determination of UTP and ATP pool sizes in human tonsillar lymphocytes by using Escherichia coli RNA polymerase. 109 46

A method was devised for directing RNA polymerase on a single promoter site on T7 DNA. Initiation complexes were formed on each of the three main promoter sites using one dinucleotide plus one nucleoside triphosphate. The ternary initiation complexes are resistant to rifampicin action, to inhibition by (rI)n at 0 degrees C and are stable at high salt concentrations. A minimum of a trinucleotide is required to form a stable ternary complex. To determine which promoter site was selected by RNA polymerase during initiation, the (rI)n-resistant RNA was digested by RNAse III to generate three characteristic initiator RNA fragments, resolved by gel electrophoresis. The three major promoter sites could be selected individually by using different primer and substrate combinations ApC plus ATP selected promoter A3, CpG plus CTP selected A2 and CpC plus ATP specified preferentially A1. A number of primer-substrate combinations specified each site at low salt concentration but the substrate requirement became very stringent at high salt concentration, suggesting that the postulated local opening of the promoter site could be more or less extensive, depending on the ionic strength. The minimum opening observed at high salt concentration corresponded to the insertion of a leader trinucleotide sequence. The promoter region melted by RNA polymerase at low salt concentration was (G plus C)-rich and corresponded to about 9 to 11 base pairs. Sequences of the melting recognition regions were tentatively inferred from the results.
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PMID:Interaction of RNA polymerase from Escherichia coli with DNA. Analysis of T7 DNA early-promoter sites. 110 Apr 9

The kinetics of the RNA chain initiation reaction carried out by RNA polymerase bound to the initiator region of a DNA template have been analyzed. Initiation proceeds in a two-substrate reaction in which the initial binary complex (enzyme-DNA) is transformed into a ternary complex (enzyme-DNA-RNA) by formation of a dinucleoside tetraphosphate and release of inorganic pyrophosphate. In this reaction RNA polymerase serves as a reactant rather than acting catalytically. The concentration of the reacting binary complex decreases throughout the reaction; hence steady state approximations cannot be used. Kinetic equations for an ordered two-substrate reaction are derived. These are most useful for the special case of reaction in the presence of an inhibitor of initiation, such as rifampicin. Equations for the latter instance are solved exactly with recourse to the steady state approximation. It is found that measurements of the extent of the initiation reaction determined at different inhibitor and substrate concentrations can give information about the initiation reaction analogous to that obtained in standard steady state kinetic analysis. This theory is applied to the experimental study of the initiation reaction carried out by Escherichia coli RNA polymerase. It is found that the inhibitor rifampicin, which blocks the initiation reaciton, acts by binding to the same form of the binary complex as the nucleoside triphosphate substrate (ATP or GTP) which is incorporated into the 5' terminus of nascent RNA molecule. The binding of the 5'-terminal nucleoside triphosphate to the enzyme appears to be rate-limiting for the initiation reaction under standard assay conditions. Initiation appears to follow an ordered reaction mechanism; however, the order of addition of the two substrates is still uncertain.
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PMID:Kinetic analysis of ribonucleic acid chain initiation by Escherichia coli Ribonucleic acid polymerase bound to DNA. 110 16

Infection of Pseudomonas putida by the bacteriophage gh-L-induced the synthesis of a novel DNA-dependent RNA polymerase. This gh-L-induced RNA polymerase was purified to near homogeneity. It was shown to be distinct from the host RNA polymerase (alpha-2 beta beta sigma) physically and in respect to many of its catalytic properties. The gh-L-induced RNA polymerase was composed of a single polypeptide of approximately 98,000 molecular weight. The divalent metal ion requirement for in vitro RNA synthesis by the gh-L-polymerase could be satisified with Mg-2+, but not with Mn-2+. Rna synthesis by the gh-L polymerase was highly resistant to inhibition by rifampicin and streptolydigin but could be inhibited by relatively low concentrations of KCl or the rifamycin derivative AF/013. The structural analog of ATP, 3'-deoxyadenosine 5'-triphosphate, inhibited both the gh-L-induced and the host RNA polymerases by competing for a single binding site with ATP. The phage polymerase was extremely sensitive to this inhibitor, exhibiting an apparent K-i value (2 times 10-8 M) approximately 100 times lower than that for the host RNA polymerase. The gh-L polymerase had a highly specific template requirement for DNA from the homologous gh-L phage. It would not efficiently utilize denatured DNA templates and had only low levels of activity with pyrimidine-containing polydeoxyribonucleotide homopolymers.
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PMID:Purification and characterization of bacteriophage gh-I-induced deoxyribonucleic acid-dependent ribonucleic acid polymerase from Pseudomonas putida. 111 26

A procedure has been developed for the purification of soluble DNA-dependent RNA polymerase (EC 2.7.7.6) from rye embryos. The enzyme solubilized by high salt extraction with sonication and resolved by DEAE-cellulose chromatography yields two activities. Enzyme I eluted at 0.15 M (NN4)2SO4, was insensitive to alpha-amanitin and was extremely labile. Enzyme II eluted at 0.25 M (NH4)2SO4 was inhibited by alpha-amanitin. However, DEAE-Sephadex chromatography yields three DNA-dependent RNA polymerases. Enzyme I is resistant to amanitin, while II and III enzymes are inhibited by this poison. Partially purified on DEAE-cellulose, polymerase II was further purified by hydrophobic chromatography on an omega-aminobutyl-Sepharose column. After omega-aminobutyl-Sepharose chromatography, enzyme II was stable and was more active with denatured than with native DNA as template. The activity of purified RNA polymerase II is dependent on the DNA, Mn-2+ and Mg-2+ added and requires ATP, GTP, CTP and UTP for its maximum activity. Transcription is inhibited besides by alpha-amanitin, by chromomycin A3, daunomycin, ethidium bromide and actinomycin D. Rifampin and rifamycin SV do not inhibit the enzyme. Synthetic copolymers were also effective as templates.
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PMID:Isolation and purification of RNA polymerases from rye embryos. 112 11

Factors affecting the inhibition of RNA polymerase II from rat liver by the O-n-octyloxime of 3-formylrifamycin SV (AF/013) were investigated. Using either native or denatured calf-thymus DNA as template, almost complete inhibition of RNA polymerase II was observed when AF/013 was added directly to the enzyme. Considerable resistance to AF/013 was observed when RNA polymerase II was preincubated with denatured DNA at either 0 or 37 degrees. However, under similar conditions, no resistance was observed when enzyme was preincubated with native DNA. Only when AF/013 was added to the ongoing reaction using native DNA did a resistance to AF/013 occur. The inhibition of RNA polymerase II by AF/013 was competitive with respect to all four nucleoside triphosphate substrates. The inhibition by AF/013 remaining after enzyme-DNA complex formation also appeared competitive with nucleoside triphosphate levels. The effect of exogenous protein (bovine serum albumin, BSA) on the inhibition of RNA polymerase II was also investigated. BSA reduced the extent of inhibition by AF/013, but did not alter the competitive nature of inhibition. Concurrently, the inhibition of highly purified nuclear poly(A) polymerase from rat liver, a template independent enzyme which incorporates AMP in a chain elongation reaction, was examined. As in the case of RNA polymerase, poly(A) polymerase was inhibited by AF/013 in a manner competitive with the nucleoside triphosphate substrate. The competitive nature of inhibition of RNA polymerase by AF/013 with respect to all four nucleoside triphosphate substrates, before and after enzyme-DNA complex formation, as well as the competitive nature of inhibition of poly(A) polymerase with respect to ATP tend to indicate that the major effect of AF/013 on RNA polymerase II is at the level of the substrate binding as opposed to a specific inhibition of initiation.
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PMID:Mechanism of inhibition of RNA polymerase II and poly(adenylic acid) polymerase by the O-n-octyloxime of 3-formylrifamycin SV. 116 99

1. Nucleolar RNA polymerase Ib obtained from auxin-treated lentil roots exhibits a higher transcriptional activity than the enzyme obtained from control roots. This difference is due to a change in the enzyme properties after auxin treatment. It is suggested that the hormonal effect is mediated by a factor that changes the molecular properties of nucleolar RNA polymerase. 2. Four fractions, alpha, beta, gamma and delta, that stimulate the activity of RNA polymerase Ib, have been extracted from lentil roots. Two of them, gamma and delta have been studied. Factor delta can stimulate nucleolar polymerase Ib and the nucleoplasmic enzyme II equally well, while factor gamma is specific for polymerase Ib. 3. The curve of UMP incorporation in vitro, with and without factors gamma or delta suggests that they are initiation factors. This conclusion is reinforced by the analysis of simultaneous incorporation of [gamma-32P]ATP and [3H]UMP in the RNAs synthesized in vitro. 4. Although the level of factor delta is independent of auxin treatment, that of factor gamma is doubled in auxin-treated roots. These results suggest that factor gamma is an auxin-induced protein that modulates the specific activity of the nucleolar RNA polymerase. 5. A general model of the mode of action of auxins at the molecular level is proposed. It integrates into a unified scheme the above results as well as those obtained by other workers.
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PMID:Hormonal control of transcription in higher plants. 116 23

A nuclear poly(A) polymerase has been isolated from oviducts of immature quails. It could be purified 4300-fold. The enzyme depends specifically on ATP as substrate and requires Mg2+. The most effective primer for the enzyme is a polynucleotide, isolated from oviduct tissue. A poly(A) sequence to a maximum of 60 AMP residues is covalently linked per primer molecule. The poly(A)-rich product of the enzymatic reaction can be annealed to oligo(dT)-cellulose. The purest fraction does not contain any detectable poly(A)-degrading enzyme activity. Only very low activities of RNA polymerase are present. The poly(A polymerase activity in the assay with ATP is reduced by the ATP analogue, beta, lambda-ATP-methylene-diphosphonate. Both K-m and V are lowered. The ATP analogue is incorporated to a smaller extent into the poly(A) sequence, synthesized by the enzyme. Several other analogues of adenine, adenine nucleosides and adenine nucleotides are without effect on the enzymatic reaction. By these properties poly(A) polymerase can be distinguished from RNA polymerases form I and form II, isolated from the same tissue. Actinomycin D and alpha-amanitin failed to inhibit poly(A) polymerase activity. The activity of poly(A) polymerase has been determined during primary stimulation with the estrogen analogue diethylstilbestrol (daily injection for 5 days), after withdrawal of the hormone for 17 days and after secondary stimulation with the hormone analogue. The enzyme activity does not change during primary stimulation, withdrawal of the hormone or secondary stimulation. However the activity of a poly(A) degrading enzyme, localized in the nucleus, is reduced in oviducts from hormone-treated quails.
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PMID:Poly(A) polymerase in quail oviduct. Changes during estrogen induction. 116 81

Under specific binding conditions RNA polymerase forms complexes at several sites of the replicative form DNA of bacteriophage fd. One of these complexes becomes stable to both high salt and low temperature after incubation with GTP. None of the complexes is stabilized by ATP. The stabilization by GTP results from the synthesis of an oligo(G) chain, which is bound in the complex. Size and pyrimidine fingerprints of the DNA segment protected by the enzyme against digestion with DNase are not changed upon initiation of oligo(G) synthesis. This result indicates that binding site and initiation site are identical parts of a promoter region.
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PMID:Stabilization of promoter complexes with a single ribonucleoside triphosphate. 117 37

A non-histone protein has been isolated from Ehrlich ascites tumor chromatin. The minimum molecular weight of this non-histone protein, estimated by sodium dodecyl sulfate gel electrophoresis and amino acid analysis, is approximately 10 to 11,000. This non-histone protein is acidic, contains 2.7% alkalilabile phosphorus, binds to DNA, and inhibits transcription of DNA in vitro by the homologous RNA polymerase. The per cent inhibition of RNA synthesis is not affected by increasing amounts of RNA polymerase, but is reduced by addition of excess DNA. In the presence of the non-histone protein, incorporation of [gamma-32P]ATP into RNA in the in vitro RNA synthesizing system is inhibited, with no apparent change in the average chain length of the RNA product. Inhibition of RNA synthesis is completely eliminated if the DNA template is allowed to interact with ATP prior to the addition of the non-histone protein. These results indicate that the observed repression of in vitro RNA synthesis is due to the effect of the non-histone protein on the DNA, inhibiting the initiation of RNA chain formation.
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PMID:Inhibition of transcription in vitro by a non-histone protein isolated from Ehrlich ascites tumor chromatin. 119 69


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