Animal DNA-Dependent RNA Polymerases. 1. Large-Scale Solubilization and Separation of A and B Calf-Thymus RNA-Polymerase Activities

Abstract A procedure is described for obtaining DNA-dependent RNA polymerase form B from calf thymus in high purity. The procedure includes homogenization in low salt, sedimentation of chromatin at 50 000 x g, adsorption to DEAE-cellulose in a batch procedure, DNA-agarose chromatography, and chromatography on DEAE-Sephadex. Analyses of the preparation by band sedimentation and gel-electrophoresis indicate a high purity of the enzyme.

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Novel insertion and deletion mutants of RpoB that render Mycobacterium smegmatis RNA polymerase resistant to rifampicin-mediated inhibition of transcription

Microbiology ◽

10.1099/mic.0.036970-0 ◽

2010 ◽

Vol 156 (5) ◽

pp. 1565-1573 ◽

Cited By ~ 11

Author(s):

Vidyasagar Malshetty ◽

Krishna Kurthkoti ◽

Arnab China ◽

Bratati Mallick ◽

Subburaj Yamunadevi ◽

...

Keyword(s):

Rna Polymerase ◽

Mycobacterium Smegmatis ◽

Three Dimensional ◽

Rna Polymerases ◽

Dimensional Structure ◽

Deletion Mutants ◽

Insertion And Deletion ◽

Calf Thymus ◽

Specific Activities ◽

Spatial Positioning

The startling increase in the occurrence of rifampicin (Rif) resistance in the clinical isolates of Mycobacterium tuberculosis worldwide is posing a serious concern to tuberculosis management. The majority of Rif resistance in bacteria arises from mutations in the RpoB subunit of the RNA polymerase. We isolated M. smegmatis strains harbouring either an insertion (6 aa) or a deletion (10 aa) in their RpoB proteins. Although these strains showed a compromised fitness for growth in 7H9 Middlebrook medium, their resistance to Rif was remarkably high. The attenuated growth of the strains correlated with decreased specific activities of the RNA polymerases from the mutants. While the RNA polymerases from the parent or a mutant strain (harbouring a frequently occurring mutation, H442Y, in RpoB) were susceptible to Rif-mediated inhibition of transcription from calf thymus DNA, those from the insertion and deletion mutants were essentially refractory to such inhibition. Three-dimensional structure modelling revealed that the RpoB amino acids that interact with Rif are either deleted or unable to interact with Rif due to their unsuitable spatial positioning in these mutants. We discuss possible uses of the RpoB mutants in studying transcriptional regulation in mycobacteria and as potential targets for drug design.

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Cibacron Blue inhibition of prokaryotic and eukaryotic DNA-dependent RNA polymerases

Collection of Czechoslovak Chemical Communications ◽

10.1135/cccc19902769 ◽

1990 ◽

Vol 55 (11) ◽

pp. 2769-2780

Author(s):

Aleš Cvekl ◽

Květa Horská

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Wheat Germ ◽

Rna Polymerase Iii ◽

Enzymic Activity ◽

Rna Polymerases ◽

Cibacron Blue ◽

E Coli ◽

Polymer Formation ◽

Calf Thymus

A comparison was drawn between the action of Cibacron Blue F3GA on the enzymic activity of DNA-dependent RNA polymerases from different sources, e.g. Escherichia coli, calf thymus and wheat germ (polymerase II). Sensitivity towards this inhibitor was determined for polymer formation and primed abortive synthesis of trinucleotide UpApU. In case of E. coli polymerase and wheat germ polymerase II the dye inhibits both polymer formation and abortive synthesis. Calf thymus polymerase II is inhibited only in the polymerisation step. The primed initiation reaction was found to be resistant towards the dye. In case of E. coli polymerase and wheat germ polymerase II the sensitive step is the formation of internucleotide bond whereas in case of calf thymus polymerase II the translocation of the enzyme is influenced. An analysis of kinetic data indicates more than one binding site for the dye on RNA polymerase II from calf thymus and wheat germ. Cibacron blue does not inhibit specific transcription catalyzed by RNA polymerase III from human HeLa cells and mouse leukemia L1210 cells.

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STRUCTURAL AND FUNCTIONAL PROPERTIES OF THREE MAMMALIAN NUCLEAR DNA-DEPENDENT RNA POLYMERASES

Acta Endocrinologica ◽

10.1530/acta.0.071s222 ◽

1972 ◽

Vol 71 (2_Suppla) ◽

pp. S222-S246 ◽

Cited By ~ 23

Author(s):

P. Chambon ◽

F. Gissinger ◽

C. Kedinger ◽

J. L. Mandel ◽

M. Meilhac ◽

...

Keyword(s):

Molecular Weight ◽

Rna Polymerase ◽

Rat Liver ◽

Nuclear Dna ◽

Active Role ◽

Rna Polymerases ◽

E Coli ◽

Structural And Functional Properties ◽

Calf Thymus ◽

Derivatives Of

ABSTRACT Three DNA-dependent RNA polymerases AI, BI and BII were purified from calf thymus. These enzymes were characterized by their sensitivity to amanitin, their subunit pattern and their template specificities. Enzyme AI is resistant to amanitin, while B enzymes are inhibited by this poison. Each of these enzymes contains two subunits of high molecular weight: A1 (M.W. 197 000) and A2 (M.W. 126 000) for enzyme AI, Bl (M.W. 214 000) and B3 (M.W. 140 000) for enzyme BI, B2 (M.W. 180 000) and B3 for enzyme BII. In addition, each enzyme contains several subunits of lower molecular weight. Purification of B enzymes from rat liver showed the presence of two enzymes similar to calf thymus RNA polymerases BI and BII and suggested the existence of an additional B enzyme in rat liver. The number of RNA polymerase form B molecules was determined in a variety of animal tissues by binding of labelled amanitin. The values ranged from 1 × 104 to 6.5 × 104 molecules per haploid genome. Studies with derivatives of rifamycin indicated that calf thymus B enzymes were able to recognize specific initiation sites on calf thymus DNA, which were different from those recognized by enzyme AI or E. coli RNA polymerase. These results favour the hypothesis that the multiplicity of animal RNA polymerases could play an active role in the control of transcription.

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Degree of inhibition of eukaryotic DNA-dependent RNA polymerases by thuringiensin

Collection of Czechoslovak Chemical Communications ◽

10.1135/cccc19810673 ◽

1981 ◽

Vol 46 (3) ◽

pp. 673-677

Author(s):

Květa Horská ◽

Karel Šebesta

Keyword(s):

Rna Polymerases ◽

Calf Thymus ◽

Eukaryotic Dna

It was shown in experiments with purified DNA-dependent RNA polymerases from calf thymus that the inhibition of these enzymes caused by thuringiensin is competitive with respect to ATP. Enzyme AI is more sensitive to the inhibitor (Ki = 0.34 μm) than enzyme B (Ki = 1.49 μm).This finding eliminates the discrepancies with respect to this subject which exist in literature.

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