scholarly journals The mechanism of inhibition of ribonucleic acid synthesis by 8-hydroxyquinoline and the antibiotic lomofungin.

1975 ◽  
Vol 147 (3) ◽  
pp. 401-410 ◽  
Author(s):  
R S Fraser ◽  
J Creanor
Keyword(s):  
Rna Polymerase ◽  
Direct Contact ◽  
Rna Synthesis ◽  
Chelating Agent ◽  
Acid Synthesis ◽  
Polymerase Activity ◽  
5S Rna ◽  
Bivalent Cations ◽  
Mechanism Of Inhibition ◽  
Dna Template

RNA synthesis in yeast is rapidly inhibited by 8-hydroxyquinoline and the phenazine antibiotic lomofungin (5-formyl-1-methoxycarbonyl-4,6,8-trihydroxyphenazine). It is shown that lomofungin, like 8-hydroxyquinoline, is a chelating agent for bivalent cations. The mechanism of inhibition of RNA synthesis by lomofungin and 8-hydroxyquinoline was investigated in experiments with isolated Escherichia coli RNA polymerase. The results show that both inhibitors are capable of inhibiting polymerase activity solely by chelating the dissociable cations Mn2+ and Mg2+. Evidence is presented which shows that inhibition may occur in the absence of any direct contact between the RNA polymerase or DNA template and the inhibitor. The possibility that inhibition might also occur by chelation of the Zn2+, which is tightly bound to the polymerase, is discussed: it is concluded that lomofungin or 8-hydroxyquinoline is likely to inhibit the enzyme by removal of Mn2+ and Mg2+ before chelating the Zn2+. On the basis of inhibition by chelation of Mn2+ and Mg2+, explanations are proposed for why lomofungin and 8-hydroxyquinoline inhibit synthesis of ribosomal and polydisperse RNA more than that of 5S RNA and tRNA, and for why protein synthesis is not immediately inhibited in the intact yeast cell.

scholarly journals Mechanism of inhibition of Escherichia coli RNA polymerase by captan

1982 ◽  
Vol 201 (1) ◽  
pp. 145-151 ◽  
Author(s):  
J W Dillwith ◽  
R A Lewis
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Rna Synthesis ◽  
Polymerase Activity ◽  
Mechanism Of Inhibition ◽  
Dna Binding Site ◽  
Rna Polymerase Activity ◽  
Dna Template ◽  
Template Dna

Captan (N-trichloromethylthiocyclohex-4-ene-1,2-dicarboximide) was shown to inhibit RNA synthesis in vitro catalysed by Escherichia coli RNA polymerase. Incorporation of [gamma-32P]ATP and [gamma-32P]GTP was inhibited by captan to the same extent as overall RNA synthesis. The ratio of [3H]UTP incorporation to that of [gamma-32P]ATP or of [gamma-32P]GTP in control and captan-treated samples indicated that initiation was inhibited, but the length of RNA chains being synthesized was not altered by captan treatment. Limited-substrate assays in which re-initiation of RNA chains did not occur also showed that captan had no effect on the elongation reaction. Studies which measured the interaction of RNA polymerase with template DNA revealed that the binding of enzyme to DNA was inhibited by captan. Glycerol-gradient sedimentation of the captan-treated RNA polymerase indicated that the inhibition of the enzyme was irreversible and did not result in dissociation of its subunits. These data are consistent with a mechanism in which RNA polymerase activity was irreversibly altered by captan, resulting in an inability of the enzyme to bind to the template. This interaction was probably at the DNA-binding site on the polymerase and did not involve reaction of captan with the DNA template.

scholarly journals RNA Polymerase Activity Assay on Biochips: Correlation between Template DNA Density and RNA Synthesis

2010 ◽  
Vol 31 (7) ◽  
pp. 2107-2109 ◽  
Author(s):  
Bok-Hui Lee ◽  
Hyun-Jung Seo ◽  
So-Hyun Kim ◽  
Woong Jung ◽  
Dong-Woon Kim ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Synthesis ◽  
Polymerase Activity ◽  
Activity Assay ◽  
Rna Polymerase Activity ◽  
Template Dna ◽  
Dna Density

scholarly journals Deoxyribonucleic acid-dependent ribonucleic acid polymerases from murine spleen cells. Increased amounts of the nucleolar species in leukaemic tissue

1973 ◽  
Vol 133 (4) ◽  
pp. 797-804 ◽  
Author(s):  
Donner F. Babcock ◽  
Marvin A. Rich
Keyword(s):  
Rna Synthesis ◽  
Rna Polymerases ◽  
Infected Tissue ◽  
Leukaemia Virus ◽  
Bivalent Cations ◽  
Murine Spleen ◽  
Insoluble Product ◽  
Polymerase I ◽  
Dna Template ◽  
Nucleolar Rna

1. In the spleens of infected mice, the Friend leukaemia virus induces a sharp increase in the ability of subsequently isolated nuclei to incorporate exogenous UTP into an acid-insoluble product. Inhibitor studies indicate that the incremental RNA synthesis proceeds from a DNA template and that both nucleolar and nucleoplasmic activities are involved. 2. The partially purified DNA-dependent RNA polymerases from control and virus-infected tissue are indistinguishable with respect to chromatographic mobility, dependence on bivalent cations, ionic strength, pH and their susceptibility to α-amanitin. The RNA polymerases of the murine spleen resemble the enzymes of other mammalian tissue in these properties. 3. A comparison of the amount of polymerase solubilized from normal and infected tissue correlates with the activity observed in assays of the respective nuclei. These experiments indicated that the increase in nucleolar RNA synthesis after infection is mediated by increased extractable polymerase I activity whereas the change in nucleoplasmic RNA synthesis results from an alteration of chromatin or a chromatin-associated factor.

scholarly journals Decreased ribonucleic acid synthesis in isolated rat liver nuclei during starvation

1970 ◽  
Vol 120 (2) ◽  
pp. 381-384 ◽  
Author(s):  
D. Rickwood ◽  
H. G. Klemperer
Keyword(s):  
Rna Polymerase ◽  
Ammonium Sulphate ◽  
Ribonucleic Acid ◽  
Rna Synthesis ◽  
Actinomycin D ◽  
Acid Synthesis ◽  
Isolated Nuclei ◽  
Isolated Rat Liver ◽  
Liver Nuclei ◽  
Isolated Rat

1. Isolated nuclei from starved rats showed a lowered incorporation of [14C]UMP into RNA. 2. The Mg2+-dependent incorporation was decreased by 30% after 1 day of starvation, but incorporation in the presence of Mn2+ and ammonium sulphate decreased only after longer periods of starvation. 3. RNA synthesis by nuclei in the presence of excess of added RNA polymerase was unchanged after 1 day of starvation and was inhibited by 20% after 4 days. 4. The capacity of nuclei to bind actinomycin D was unchanged after 1 day and was decreased by 20% after 4 days of starvation.

In vitro transcription of RNA in nuclei, nucleoli and chromatin from physarum polycephalum

1977 ◽  
Vol 26 (1) ◽  
pp. 267-279
Author(s):  
K.E. Davies ◽  
I.O. Walker
Keyword(s):  
Rna Polymerase ◽  
Physarum Polycephalum ◽  
Rna Synthesis ◽  
In Vitro Transcription ◽  
Polymerase Activity ◽  
Controlled Conditions ◽  
Rna Polymerase Activity ◽  
Alpha Amanitin

Methods for isolating nuclei, nucleoli and chromatin from Physarum polycephalum which retain high levels of endogenous RNA polymerase activity are described. Under carefully controlled conditions with respect to mono- and divalent cation concentrations RNA synthesis in nuclei displayed linear kinetics for at least 30 min and the RNA products had a similar size distribution to nuclear RNA synthesis observed in vivo. Chromatin showed 60% of the nuclear transcriptional activity but no conditions were found where faithful transcription of the template occurred. Isolated nucleoli were 5-fold more active than nuclei and the endogenous RNA polymerase activity was insensitive to alpha-amanitin. Under carefully controlled conditions, the nucleoli appeared to support the accurate transcription, re-initiation and processing of rRNA chains in vitro.

Mammary Gland Nuclear RNA Polymerase Activities in Pregnant, Lactating, and 7,12-Dimethylbenz(α)anthracene-Induced Mammary Tumor-Bearing Rats

1972 ◽  
Vol 50 (6) ◽  
pp. 644-653 ◽  
Author(s):  
I. S. Mendelson ◽  
K. M. Anderson
Keyword(s):  
Mammary Gland ◽  
Ionic Strength ◽  
Rna Polymerase ◽  
Ammonium Sulfate ◽  
Sea Urchin ◽  
Rat Liver ◽  
Rna Synthesis ◽  
Polymerase Activity ◽  
Differential Sensitivity ◽  
Nuclear Rna

RNA synthesis catalyzed by nuclei isolated from mammary glands of pregnant and lactating rats and from 7,12-dimethylbenz(α)anthracene-induced mammary tumors was examined, and the following observations were made.(1) Assay 1 (6 mM Mg2+, low ionic strength, nucleolar polymerase I), and assay 3 (2 mM Mg2+, 1.6 mM Mn2+, 0.3 M ammonium sulfate, high ionic strength, nucleoplasmic polymerase II) promoted synthesis of ribosomal-like (rRNA) and nonribosomal-like RNA (dRNA), respectively, as verified by differential sensitivity to actinomycin D, double-labelling experiments, and response to α-amanitin.(2) Synthesis of a rRNA-like product was increased in assay 2 (6 mM Mg2+, 0.02 M ammonium sulfate), compared with assay 1; in assay 3 increased formation of a more dRNA-like product occurred. Stimulation of mammary gland nuclear RNA synthesis by ammonium sulfate is biphasic, similar to the response of rat liver and sea urchin nuclei.(3) In assay 1, more rRNA was synthesized at 30° than at 37°; in assay 3 somewhat greater synthesis of dRNA occurred at 37° compared with 30°. The pH optima in assays 1 and 3 of 8.0 and 8.5, respectively, are the opposite of those reported for rat liver nuclei.(4) During pregnancy, isolated nuclei synthesized more dRNA-like product, compared to lactation; nuclei from lactating glands formed more rRNA, than during pregnancy. Nuclei from slowly growing tumors (Ts) examined in the three assays were 30–50% as active, while those from rapidly growing tumors (Tf) exceeded the activities of nuclei from pregnant (P) or lactating (L) rats.(5) P, L, and Ts nuclei incubated in assay 3 with α-amanitin were inhibited about 60%, compared to an 80% reduction with nuclei from tumors that were growing rapidly. The ratio of the base G to either A or U did not return to that of assay 1 or 2 (rRNA). This result is consistent with the presence of a third nucleoplasmic RNA polymerase (enzyme "III"), as described with rat liver and sea urchin nuclei.(6) Nuclei from proliferating normal and neoplastic tissues exhibited greater polymerase II activity, and the ratio of nucleoplasmic to nucleolar enzyme activity was increased. Changes in activities of enzyme II and enzyme "III" do not appear to be necessarily coordinate.(7) The pattern of polymerase activity in P and L nuclei is probably related to cellular proliferation and/or hypertrophy during pregnancy, and cellular function represented by milk protein synthesis with lactation. RNA polymerase activity in T nuclei correlated with the growth rate of the parent tumor and not with its histology, at least not in any simple way.

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