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 antibiotic rifampicin forms a very tight complex with DNA-dependent RNA polymerase of Escherichia coli. The rate constants of association and dissociation of this complex have been measured and found to be dependent on the purity of the enzyme. Thus a crude RNA polymerase (fraction-3 enzyme) has rate constants different from those of an enzyme further purified by DEAE-cellulose chromatography (fraction-4 enzyme). The complex produced by the antibiotic and the fraction-3 enzyme is about ten times more stable and is formed about ten times more slowly than the complex with fraction-4 enzyme. It has been shown that the RNA present in the crude enzyme and removed by chromatography on DEAE-cellulose is the cause of the change in the kinetics of the complex. tRNA of rat liver and crude rat liver RNA added to purified RNA polymerase have a similar effect. Mg2+, which has no intrinsic influence, augments the effect of the nucleic acids, whereas monovalent cations do not. Since nucleic acids increase the stability of the complex, but at the same time decrease the rat of its formation, the equilibrium constant, Keq, remains almost the same. The possible effects of nucleic acids on the rifampicin binding site are discussed.
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PMID:On the kinetics of the rifampicin-RNA-polymerase complex. Differences between crude and purified enzyme fractions. 78 Jan 10

Studies were undertaken to understand the control of synthesis, stability and modification of UDP galactose epimerase and DNA-dependent RNA polymerase during sporulation of Saccharomyces cerevisiae. When a pre-induced culture of an inducible strain (wild type) is transferred to sporulation medium, the epimerase is inactivated to an undetectable level within 16 hours. Surprisingly, the addition of cycloheximide, a protein synthesis inhibitor, during sporulation stabilizes the epimerase activity. However, in a constitutive strain, the epimerase continues to be synthesized de novo during sporulation. Since the enzyme is synthesized during both vegatative growth and sporulation constitutively, the controls for synthesis of epimerase must be similar under these physiologically different conditions. After chromatography on DEAE Sephadex, there is no change observed in the elution patterns of RNA polymerase forms extracted from acetate growth vegetative cells, sporulating cells or from mature asci ; in all cases RNA polymerase consists of three forms, Ib, II and III. However, single spore suspension obtained from asci by treatment with zymolase contains a new form with chromatographic properties similar to those of form Ia. Our data suggests that form Ia may be a modification product of from Ib.
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PMID:Control of enzyme synthesis and stability during sporulation in Saccharomyces cerevisiae. 78 57

Three peaks of DNA-dependent RNA polymerase (EC 2.7.7.6) activity were resolved when the enzyme was prepared from the isolated macronuclei of Tetrahymena pyriformis GL(amicronucleate strain) and chromatographed on DEAE-Sephadex A25. They were eluted at around 0.05, 0.15, and 0.2 M of ammonium sulfate, and termed TIa, TIb, and TII, respectively. All three enzymes transcribed heat-denatured DNA more efficiently, especially the peak TII, detecable only when heat-denatured DNA was used as a template. Further characterization of each enzyme, after they were rechromatographed on DEAE-Sephadex, demonstrated the similarity in many respects of TIa and TIb, and the distinct nature of the TII enzyme. TIa, TIb, and TII were all insensitive to rifampicin, while only TII was substantially inhibited by alpha-amanitin. On the other hand, the activity of TII was progressively lowered by increasing the concentration of ammonium sulfate in the assay mixture, a finding incompatible with those obtained thus far. It is concluded from the data that the Tetrahymena polymerase is of eukaryotic and not of bacterial type in spite of the findings indicating the bacterial nature of this organism.
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PMID:DNA-dependent RNA polymerase from a protozoan, Tetrahymena pyriformis. Extraction and partial characterization. 80 68

A new form of DNA-dependent RNA polymerase termed enzyme III has been purified from sporulating cells of Bacillus subtilis. In addition to the subunits of core RNA polymerase (beta', beta, alpha, and omega), enzyme III contains sporulation-specific polypeptides of 85,000 (P85) and 27,000 (P27) daltons. P85 corresponds to an RNA polymerase-binding protein previously identified by precipitation of RNA polymerase from crude extracts of sporulating cells with antibody directed against core enzyme. Both P85 and P27 co-purified with RNA polymerase highly purified by gel filtration, DEAE-cellulose chromatography, phosphocellulose chromatography, and glycerol gradient centrifugation. Enzyme III bound more tightly to phosphocellulose and sedimented more rapidly during zone centrifugation than did RNA polymerase lacking the sporulation polypeptides. RNA polymerase containing P85 and P27 transcribed B. subtilis DNA about 4.5 times more actively than did core RNA polymerase, although both enzymes exhibited similar activities with poly(dA-dT) and phage phie DNA as templates. Enzyme III and core RNA polymerase also differed in their response to increasing concentrations of Mg2+ and KCl.
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PMID:RNA polymerase from sporulating Bacillus subtilis. Purification and properties of a modified form of the enzyme containing two sporulation polypeptides. 81 62

The activities of RNA polymerases I and II have been measured in 3T6 during the transition from the resting to growing state by solubilization of the enzymes followed by chromatography on DEAE-Sephadex columns. The activity of RNA polymerase II remains unchanged during the first 12 h after serum stimulation while the activity of RNA polymerase I increases and closely parallels the increased activity seen in isolated nuclei. Compared to enzyme from resting cells. RNA polymerase I from serum stimulated cells elutes at a lower ammonium sulfate concentration on DEAE-Sephadex chromatography and its activity shows distinctly different dependencies on the concentration of ammonium sulfate and magnesium ion. These observations are discussed in relation to the possible mechanism by which 3T6 regulates the synthesis of preribosomal RNA.
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PMID:Solubilized DNA-dependent RNA polymerase activities in resting and growing fibroblast. 83 13

DNA-dependent RNA polymerases were extracted from rat uterine tissue, partially purified and resolved by DEAE-Sephadex chromatography. RNA polymerases I, II, IIIA, and IIIB eluted at the characteristic ammonium sulfate concentrations of 0.15, 0.28, 0.34, and 0.42 M, respectively. The sensitivity of each peak of polymerase to alpha-amanitin was examined and was shown to be essentially identical to the three classes of RNA polymerases in other mammalian systems. RNA polymerase I was insensitive to high levels of alpha-amanitin, RNA polymerase II was sensitive to low concentrations of alpha-amanitin (50% inhibition at 0.006 mug/ml) and RNA polymerases IIIA and IIIB were sensitive to high concentrations of alpha-amanitin (50% inhibition at 18 mug/ml). The alpha-amanitin sensitivity curve of total RNA synthesis measured in isolated nucleo demonstrated that the activity of each class of RNA polymerase could be quantitated in uterine nuclei. Thus the initial decrease in activity at low concentrations of alpha-amanitin (50% inhibition at 0.005 mug/ml) was attributed to the inhibition of RNA polymerase II activity, the second decrease in activity at higher concentrations of alpha-amanitin (50% inhibition at 15 mug/ml) was attributed to the inhibition of RNA polymerase III activity, and the activity which was resistant to the highest alpha-amanitin concentration tested was attributed to RNA polymerase I activity. When estradiol was given to immature rats 6 h before killing both RNA polymerases I and III levels in nuclei were increased significantly over the control values. The time course of these changes demonstrated that the increases in RNA polymerases I and III were first evident between 1.5 and 3 h following hormone treatment. Significantly, these increases in polymerase I and III in nuclei parallel the published increases for rRNA and tRNA synthesis following hormone treatment. However, the amount of RNA polymerase I and III was not altered upon extraction, suggesting that these changes are due to the alteration in chromatin template activity. Both estradiol and estriol produced identical increases in uterine RNA polymerase I and III 6 h after treatment.
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PMID:Hormonal control of transcription in the rat uterus. Stimulation of deoxyribonucleic acid-dependent RNA polymerase III by estradiol. 83 97

This paper reports the evidence for the existence of multiple sites of action of aflatoxin B1 in relation to its inhibition of rat hepatic nuclear RNA synthesis. Two hours after aflatoxin B1 injection (0.3 mg/100 g body weight), rat hepatic nuclear and nucleolar RNA synthesis, in vitro, were inhibited 70 and 90% respectively. When total nuclear free and engaged RNA polymerases were solubilized and assayed in the presence of alpha-amanitin (3.2 micrograms/ml), only alpha-amanitin-sensitive activity was reduced (50 to 70%) by aflatoxin B1. DEAE-Sephadex column chromatography confirmed this finding and further demonstrated that RNA polymerase II was the activity selectively inhibited. Since aflatoxin B1 dramatically inhibited nucleolar RNA synthesis, but had little effect on RNA polymerase I activity per se, it is concluded, therefore, that, in addition to its direct inhibitory effect on the enzymic function of RNA polymerase II, aflatoxin B1 must also cause impairment of the nucleolar DNA template function.
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PMID:Mechanism of aflatoxin B1 inhibition of rat hepatic nuclear RNA synthesis. 86 81

A simplified method is described for the large-scale preparation of a highly purified DNA-dependent RNA polymerase B from calf thymus. The method includes homogenization and lysis of the tissue, chromatography on DEAE-cellulose, phosphocellulose, hydroxylapatite-Sephadex G-10 and, once again, phosphocellulose. The procedure avoids the preparation of nuclei, the use of sonication, ammonium sulphate precipitations and dialysis steps and needs no ultracentrifugation.
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PMID:An improved method for the preparation of the DNA-dependent RNA polymerase B from calf thymus. 87 42

DNA-dependent RNA polymerase I (or A) (nucleoside triphosphate:RNA nucleotidyltransferase, EC 2.7.7.6) was purified from Ehrlich ascites cells after solubilization from isolated nuclei. The purification was accomplished by a procedure involving initial precipitation with ammonium sulfate, following by chromatographies on DEAE-Sephadex and phosphocellulose ion exchange resins and gel filtration on Sepharose 6B. A chromatographically homogeneous enzyme was obtained which was purified about 2300-fold relative to nuclear extracts. The specific activity of the most purified enzyme fraction was 230 nmol of [3H]UTP incorporated into RNA per mg of protein in 10 min at 37 degrees C, which is similar to those reported for the highly purified RNA polymerase I from mouse myeloma and calf thymus. The elution position on Sepharose 6B gel filtration indicated a molecular weight of approx. 580 000. Analysis of the purified enzyme by polyacrylamide gel electrophoresis under nondenaturing conditions revealed only one protein band. Certain heterogeneity in the RNA polymerase I fractions was found in the early chromatographic steps, but not in the most purified fractions.
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PMID:Purification and properties of RNA polymerase I from Ehrlich ascites cells. 91 51

DNA-dependent RNA polymerase III was purified from the posterior silk gland of the moth Bombyx mori by chromatography on DEAE-cellulose, DEAE-Sephadex, CM-Sephadex, and phosphocellulose and by sedimentation in sucrose density gradients. The specific activity of this chromatographically homogeneous enzyme was comparable to that reported for other purified eukaryotic RNA polymerases. Sucrose gradient sedimentation analysis suggested a molecular weight of approximately 590,000 to 660,000 for B. mori RNA polymerase III. Analysis of subunit composition by polyacrylamide gel electrophoresis under denaturing conditions showed that the chromatographically purified RNA polymerase III contained subunits with molecular weights of 155,000 (IIIa), 136,000 (IIIb), 67,000 (IIIc), 62,000 (IIId), 49,000 (IIIe), 39,000 (IIIf), 36,000 (IIIg), 31,000 (IIIh), 28,000 (IIIi), and 18,000 (IIIj). Molar ratios were close to unity for all subunits except for IIIj, which was present in an approximate molar ratio of 2. As has been observed for mammalian class III enzymes, the B. mri RNA polymerase III can be resolved into two components upon electrophoresis under nondenaturing conditions. Comparative studies of the class III enzymes from B. mori and from higher eukaryotic cells show that many of the general chromatographic and catalytic properties, as well as the overall subunit compositions, are similar for the various enzymes. However, unlike the mammalian class III enzymes, B. mori RNA polymerase III is completely resistant to high concentrations of alpha-amanitin, and it does not contain an 89,000-dalton subunit. The data are discussed in terms of the function and regulation of RNA polymerase III in lower and higher eukaryotes.
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PMID:Purification and subunit structure of deoxyribonucleic acid-dependent ribonucleic acid polymerase III from the posterior silk gland of Bombyx mori. 93 6


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