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

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.

scholarly journals RNA SYNTHESIS BY EXOGENOUS RNA POLYMERASE ON CYTOLOGICAL PREPARATIONS OF CHROMOSOMES

1973 ◽  
Vol 57 (2) ◽  
pp. 538-550 ◽  
Author(s):  
R. Sederoff ◽  
R. Clynes ◽  
M. Poncz ◽  
S. Hachtel
Keyword(s):  
Rna Polymerase ◽  
Dna Sequences ◽  
Rna Synthesis ◽  
Actinomycin D ◽  
Polytene Chromosomes ◽  
Polymerase Activity ◽  
Exogenous Rna ◽  
Rna Polymerase Activity ◽  
Chromosome Regions

Cytological preparations of Drosophila polytene chromosomes serve as templates for RNA synthesis carried out by exogenous RNA polymerase (Escherichia coli). Incorporation of labeled ribonucleoside triphosphates into RNA may be observed directly by autoradiography. Because of the effects of rifampicin, actinomycin D, ribonuclease, high salt, and the requirement for all four nucleoside triphosphates, we conclude that the labeling observed over chromosomes is due to DNA-dependent RNA polymerase activity. Using this method, one can observe RNA synthesis in vitro on specific chromosome regions due to the activity of exogenous RNA polymerase. We find that much of the RNA synthesis in this system occurs on DNA sequences which appear to be in a nondenatured state.

Inhibition of RNA polymerase from Bacillus thuringiensis and Escherichia coli by β-exotoxin

1978 ◽  
Vol 24 (5) ◽  
pp. 537-543 ◽  
Author(s):  
Donovan E. Johnson
Keyword(s):  
Escherichia Coli ◽  
Bacillus Thuringiensis ◽  
Rna Polymerase ◽  
Polymerase Activity ◽  
Growth Stages ◽  
E Coli ◽  
Synthetic Dna ◽  
Rna Polymerase Activity ◽  
Normal Sensitivity ◽  
Dna Template

The characteristics of exotoxin inhibition of deoxyribonucleic acid (DNA) dependent ribonucleic acid (RNA) polymerase isolated from Escherichia coli and Bacillus thuringiensis were investigated. RNA polymerase isolated from a variety of growth stages was partially purified and assayed using several different native and synthetic DNA templates, and exotoxin inhibition patterns were recorded for each. Although 8 to 20-h RNA polymerase extracts of E. coli retained normal sensitivity to exotoxin (50% inhibition at a concentration of 7.5 × 10−6 M exotoxin), RNA polymerase isolated from late exponential and ensuing stationary-phase cultures of B. thuringiensis were nearly 50% less sensitive than exponential RNA polymerase activity. Inhibition patterns relating culture age at the time of RNA polymerase extraction to exotoxin inhibition suggested a direct correlation between diminishing exotoxin sensitivity and sporulation. Escherichia coli RNA polymerase could be made to mimic the B. thuringiensis exotoxin inhibition pattern by removal of sigma from the holoenzyme. After passage through phosphocellulose, exotoxin inhibition of the core polymerase was 30% less than the corresponding inhibition of E. coli holoenzyme. Heterologous enzyme reconstruction and assay were not possible due to loss of activity from the B. thuringiensis preparation during phosphocellulose chromatography, apparently from the removal of magnesium. In enzyme velocity studies, inhibition with exotoxin was noncompetitive with respect to the DNA template in the RNA polymerase reaction.

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.

RNA polymerase activity in virions from Ustilago maydis

1984 ◽  
Vol 4 (1) ◽  
pp. 188-194
Author(s):  
B S Ben-Tzvi ◽  
Y Koltin ◽  
M Mevarech ◽  
A Tamarkin
Keyword(s):  
Rna Polymerase ◽  
Viral Genome ◽  
Ustilago Maydis ◽  
Polymerase Activity ◽  
Reaction Products ◽  
Double Stranded Rna ◽  
Rna Molecules ◽  
Rna Transcripts ◽  
Rna Polymerase Activity

RNA polymerase activity is associated with the double-stranded RNA virions of Ustilago maydis. The reaction products of the polymerase activity are single-stranded RNA molecules. The RNA molecules synthesized are homologous to the three classes of double-stranded RNA molecules that typify the viral genome. The single-stranded RNA synthesized is released from the virions. The molecular weight of the single-stranded RNA transcripts is about half the size of the double-stranded RNA segments, and thus, it appears that in the in vitro reaction, full-length transcripts can be obtained.

Heat stress in the presence of low RNA polymerase activity increases chromosome copy number in Escherichia coli

1988 ◽  
Vol 212 (2) ◽  
pp. 203-206 ◽  
Author(s):  
Elena C. Guzman ◽  
Alfonso Jimenez-Sanchez ◽  
Elisha Orr ◽  
Robert H. Pritchard
Keyword(s):  
Escherichia Coli ◽  
Heat Stress ◽  
Rna Polymerase ◽  
Copy Number ◽  
Polymerase Activity ◽  
Chromosome Copy Number ◽  
Rna Polymerase Activity

scholarly journals Thermolabile L-A virus-like particles from pet18 mutants of Saccharomyces cerevisiae.

1986 ◽  
Vol 6 (2) ◽  
pp. 404-410 ◽  
Author(s):  
T Fujimura ◽  
R B Wickner
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Rna Polymerase ◽  
Polymerase Activity ◽  
Nonpermissive Temperature ◽  
Wild Type ◽  
Rna Dependent Rna Polymerase ◽  
Virus Like Particles ◽  
Rna Polymerase Activity ◽  
Lines Of Evidence

pet18 mutations in Saccharomyces cerevisiae confer on the cell the inability to maintain either L-A or M double-stranded RNAs (dsRNAs) at the nonpermissive temperature. In in vitro experiments, we examined the effects of pet18 mutations on the RNA-dependent RNA polymerase activity associated with virus-like particles (VLPs). pet18 mutations caused thermolabile RNA polymerase activity of L-A VLPs, and this thermolability was found to be due to the instability of the L-A VLP structure. The pet18 mutations did not affect RNA polymerase activity of M VLPs. Furthermore, the temperature sensitivity of wild-type L-A RNA polymerase differed substantially from that of M RNA polymerase. From these results, and from other genetic and biochemical lines of evidence which suggest that replication of M dsRNA requires the presence of L-A dsRNA, we propose that the primary effect of the pet18 mutation is on the L-A VLP structure and that the inability of pet18 mutants to maintain M dsRNA comes from the loss of L-A dsRNA.

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