Purification using polyethylenimine precipitation and low molecular weight subunit analyses of calf thymus and wheat germ DNA-dependent RNA polymerase II

Biochemistry ◽  
1977 ◽  
Vol 16 (11) ◽  
pp. 2334-2343 ◽  
Author(s):  
Henry G. Hodo ◽  
Stanley P. Blatti
Keyword(s):  
Molecular Weight ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Wheat Germ ◽  
Low Molecular Weight ◽  
Calf Thymus

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

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.

Immunological analyses of selected eukaryotic RNA polymerases II

1985 ◽  
Vol 63 (12) ◽  
pp. 1217-1230 ◽  
Author(s):  
Michael F. Bettiol ◽  
Randall T. Irvin ◽  
Paul A. Horgen
Keyword(s):  
Molecular Weight ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
High Molecular Weight ◽  
Wheat Germ ◽  
Polyclonal Antibodies ◽  
Sodium Dodecyl ◽  
Cross Reactivity ◽  
Rna Polymerases ◽  
Antibody Preparations

Polyclonal antibodies to native RNA polymerase II of Achlya ambisexualis and Agaricus bisporus were produced in rabbits and in mice. Monoclonal antibodies were produced against the α-amanitin resistant RNA polymerase II of the mushroom A. bisporus. These antibodies were used in comparative cross-reactivity studies with five purified RNA polymerases II (A. bisporus, A. ambisexualis, Saccharomyces cerevisiae, wheat germ, and calf thymus). A method for quantitatively comparing cross-reactivity was developed utilizing an enzyme-linked immunosorbant assay (ELISA). ELIS A comparisons indicated that the two filamentous fungi cross-reacted effectively with one another and depending upon the preparation reacted less effectively with yeast and wheat germ RNA polymerases II. Cross-reactivity measurements were also made by immunoblotting sodium dodecyl sulfate – polyacrylamide separated RNA polymerases II. The mouse anti-A. bisporus RNA polymerase II immunoglobulin G (IgG) and the monoclonal antibody preparations did not react with high molecular subunits of A. bisporus RNA polymerase II. The sera did, however, cross-react with high molecular weight subunits of A. ambisexualis. Similarily, rabbit anti-A. ambisexualis RNA polymerase II IgG reacted only with low molecular weight subunits of A. bisporus RNA polymerase II, but reacted with high molecular weight subunits of A. ambisexualis and wheat germ. Our results indicate differences in the cross-reactivity of native and denatured RNA polymerases II and suggest differences in the tertiary and quaternary organization of the enzymes examined.

scholarly journals A DNA-dependent RNA synthesis by wheat-germ RNA polymerase II insensitive to the fungal toxin α-amanitin

1992 ◽  
Vol 285 (1) ◽  
pp. 85-90 ◽  
Author(s):  
C Job ◽  
D Shire ◽  
V Sure ◽  
D Job
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Wheat Germ ◽  
Rna Synthesis ◽  
Biochemical Properties ◽  
Size Analysis ◽  
Reaction Products ◽  
High Concentration ◽  
Fungal Toxin ◽  
Alpha Amanitin

Wheat-germ RNA polymerase II is able to catalyse a DNA-dependent reaction of RNA synthesis in the presence of a high concentration (1 mg/ml) of the fungal toxin alpha-amanitin. This anomalous reaction is specifically directed by single-stranded or double-stranded homopolymer templates, such as poly(dC) or poly(dC).poly(dG), and occurs in the presence of either Mn2+ or Mg2+ as the bivalent metal cofactor. In contrast, the transcription of other synthetic templates, such as poly(dT), poly(dA).poly(dT) or poly[d(A-T)] is completely abolished in the presence of 1 microgram of alpha-amanitin/ml, in agreement with well-established biochemical properties of class II RNA polymerases. Size analysis of reaction products resulting from transcription of (dC)n templates of defined lengths suggests that polymerization of RNA chains proceeds through a slippage mechanism. The fact that alpha-amanitin does not impede this synthetic reaction implies that the amatoxin interferes with the translocation of wheat-germ RNA polymerase II along the DNA template.

scholarly journals Effect of salts on abortive and productive elongation catalysed by wheat germ RNA polymerase II

1986 ◽  
Vol 14 (4) ◽  
pp. 1583-1597 ◽  
Author(s):  
Jacques Dietrich ◽  
Marcel Teissere ◽  
Claudette Job ◽  
Dominique Job
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Wheat Germ ◽  
Productive Elongation

scholarly journals A slow kinetic transient in RNA synthesis catalysed by wheat-germ RNA polymerase II

1988 ◽  
Vol 253 (1) ◽  
pp. 281-285 ◽  
Author(s):  
C Job ◽  
L De Mercoyrol ◽  
D Job
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Conformational Change ◽  
Wheat Germ ◽  
Rna Synthesis ◽  
Single Step ◽  
Slow Kinetic ◽  
Productive Elongation

Progress curves of U-A-primed RNA synthesis catalysed by wheat-germ RNA polymerase II on a poly[d(A-T)] template exhibit a slow burst of activity. In contrast, the progress curves of single-step addition of UMP to U-A primer in the abortive elongation reaction do not exhibit the slow burst of activity. The correlation between the kinetic transient in the productive pathway of RNA synthesis and the rate of abortive elongation is suggestive of the occurrence of a slow conformational change of the transcription complex during the transition from abortive to productive elongation. The exceptional duration of the transient burst (in the region of 4 min) may suggest a transition of a hysteretic type.

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