scholarly journals Effects of Discontinuities in the DNA Template on Abortive Initiation and Promoter Escape by Escherichia coli RNA Polymerase

2007 ◽  
Vol 282 (37) ◽  
pp. 26917-26927 ◽  
Author(s):  
Qun Wang ◽  
Thomas D. Tullius ◽  
Judith R. Levin
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Promoter Escape ◽  
Dna Template ◽  
Abortive Initiation

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 Influence of DNA Template Choice on Transcription and Inhibition of Escherichia coli RNA Polymerase

2012 ◽  
Vol 56 (8) ◽  
pp. 4536-4539 ◽  
Author(s):  
Joerg Haupenthal ◽  
Kristina Hüsecken ◽  
Matthias Negri ◽  
Christine K. Maurer ◽  
Rolf W. Hartmann
Keyword(s):  
Escherichia Coli ◽  
Drug Discovery ◽  
Rna Polymerase ◽  
Sigma Factor ◽  
Inhibitory Potency ◽  
Experimental Conditions ◽  
Early Stages ◽  
Bacterial Rna ◽  
Transcription Efficiency ◽  
Dna Template

ABSTRACTIn recent decades, quantitative transcription assays using bacterial RNA polymerase (RNAP) have been performed under widely diverse experimental conditions. We demonstrate that the template choice can influence the inhibitory potency of RNAP inhibitors. Furthermore, we illustrate that the sigma factor (σ70) surprisingly increases the transcription efficiency of templates with nonphysiological nonprokaryotic promoters. Our results might be a useful guideline in the early stages of using RNAP for drug discovery.

Partial characterization of the mode of inhibition of Escherichia coli RNA polymerase by the mixed disulfide, CoASSG

1979 ◽  
Vol 57 (4) ◽  
pp. 336-345 ◽  
Author(s):  
William C. H. Bees ◽  
Peter C. Loewen
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Equilibrium Dialysis ◽  
Mixed Disulfide ◽  
Nucleoside Triphosphates ◽  
Noncompetitive Inhibitor ◽  
Fe Complex ◽  
Short Time ◽  
Dna Template

The coenzyme A – glutathione mixed disulfide (CoASSG), when complexed with iron, is capable of inhibiting the RNA polymerase of Escherichia coli. A modified procedure involving a short time of exposure to high salt allowed the reliable preparation of CoASSG–Fe which was active in inhibiting RNA polymerase. The CoASSG–Fe complex acted as a noncompetitive inhibitor for the incorporation of all four nucleoside triphosphates but had a greater effect on GMP and CMP incorporation than AMP and UMP incorporation. Neither temperature nor ionic-strength changes affected CoASSG–Fe inhibition, and the use of rifampicin showed that CoASSG–Fe did not inhibit either the initiation or elongation processes of the polymerase. CoASSG–Fe was a more effective inhibitor at low DNA-template concentrations and it was more effective in inhibiting the incorporation of CMP and GMP on simple dG-dC containing templates and the asymmetric polymer poly d(T-C)∙poly d(G-A). The inhibition of transcription of poly d(I-C) was less effective than the inhibition of transcription of poly d(G-C). Equilibrium dialysis in microdialysis cells showed that CoASSG–Fe could associate with DNA in the absence of RNA polymerase.

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