Effect of denaturation of DNA template on activity of RNA polymerase of class B

The effect of the denaturation of homologous and calf thymus DNA on the RNA polymerase B activity purified from rat liver and spleen and Ehrlich ascites cells, was investigated in presence of either Mn2+ or Mg2+ and in presence or absence of alpha-amanitin. On the basis of the results here reported, we suggest: 1) denatured DNA is more effective than native as template for polymerase B; 2) denatured DNA template and cations might play a role in determining the extent of the reaction alpha-amanitin-polymerase B.     

Effect of distamycin a on the structure and template activity of DNA in RNA - polymerase system

It is shown that native and denatured DNA form complexes with the oligopeptide-antibiotic distamycin A. The pronounced inhibition of the incorporation of AMP into RNA in the DNA directed RNA-polymerase system is due to the interference of the antibiotic with DNA.     

Effect of estradiol on the RNA content and the activity of nucleolar RNA polymerase from rooster liver

Estradiol administration to roosters results in changes in the macromolecular composition of the liver. Besides a gradual increase in liver protein and a sudden enhancement of liver DNA after 24 hours, the most pronounced change occurs in the RNA content, viz. an increase up to 190% of controls starting 26 hours after estradiol administration.The activity of nucleolar RNA polymerase -solubilized and chromatographed on DEAE-Sephadex- is increased four-fold 26 hours after estradiol treatment. This increase was found to be due to an increased initiation frequency. Concurrently, the initiation characteristics of nucleolar RNA polymerase are changed.     

Allosteric conversion of Z DNA to an intercalated right-handed conformation by daunomycin

Absorbance and fluorescence methods were used to measure the binding of the anticancer drug daunomycin to poly (dGdC) under ionic conditions that initially favor the left-handed Z conformation of the polymer. Drug binding was cooperative under these conditions and may be fully accounted for by an allosteric model in which the drug binds preferentially (but not exclusively) to the right-handed B conformation and shifts the polymer from the Z to an intercalated right-handed conformation. Quantitative analysis of binding isotherms in terms of the allosteric model allowed for estimation of the equilibrium constants for the conversion of a base pair at a B-Z interface from the Z to the B conformation and for the formation of a base pair in the B conformation within a stretch of helix in the Z conformation. The free energy of the Z to B conversion of a base pair was calculated from this data and ranges from +0.03 to +0.3 kcal/mol over the NaCl range of 2.4-3.5 M. The free energy for the formation of a B-Z junction was nearly constant at +4.0 kcal/mol over the same range of NaCl concentrations. The salt dependence of the free energy of the Z to B transition indicates preferential Na+ binding to the Z form and that there is a net release of Na+ upon conversion of a base pair from the Z to the B conformation. The energetically unfavorable Z to B transition was found by this analysis to be driven by coupling to the energetically favorable interaction of daunomycin with B form DNA. In 3.5 M NaCl, for example, the free energy change for the overall reaction (Z DNA base pairs) + (daunomycin) in equilibrium with (right-handed complex) is -7.0 kcal/mol, nearly all of which is contributed by the binding of drug to B DNA. Analysis using the allosteric model also shows that the number of base pairs converted from the Z to the B conformation per bound drug molecule is salt dependent and provides evidence that drug molecules partition into regions of the polymer in the right-handed conformation.     

DNA-dependent RNA polymerase III from cauliflower. Characterization and template specificity

Class III DNA-dependent RNA polymerase (EC 2.7.7.6) was highly purified from cauliflower (Brassica oleracea, var. bortytis) by using polyethyleneimine precipitation. The specific activity of the enzyme was comparable to that reported for mammalian enzymes. Glycerol gradient sedimentation analysis indicated that the sedimantation coefficient (23 S) was slightly higher than that of enzyme II from cauliflower. The class III enzyme was inhibited by alpha-amanitin at high concentrations (50% inhibition at 200 microgram/ml). The Km value for nucleoside triphosphate was determined. Template specificities for single synthetic polymers showed that the enzyme read pyrimidine homopolymers as templates and preferred poly(dT) to poly(dC). The enzyme transcribed both strands of homopolymer pairs of poly(dI). poly(dC) and poly(dA).poly(dT). The synthetic polyribonucleotides were not effectively read. Competition experiments with these synthetic polymers indicated that the enzyme had different binding specificities which were not the same as their template specificities. The different binding affinities and template specificites for synthetic templates of the three classes of enzyme suggest that the enzyme can discriminate among different template sequences.     

Effect of protein phosphorylation on the activity of RNA polymerase in streptomycetes

RNA polymerase was isolated fromStreptomyces granaticolor and protein kinase was partially purified fromStreptomyces albus. When RNA polymerase was treated with protein kinasein vitro the activity of RNA polymerase was markedly enhanced. Furthermore, a protein ofM=65 kDa was isolated which, after being phosphorylated, stimulated RNA polymerase activityin vitro. Because neither the β-subunits nor the α-subunits of RNA polymerase were phosphorylated it is assumed that phosphorylation of the 65 kDa protein may regulate the activity of RNA polymerase in streptomycetes.     

Effect of phytohaemagglutinin on the nuclear RNA polymerase activity of human lymphocytes

The DNA-dependent RNA polymerase activity of isolated nuclei from human peripheral blood has been shown to increase following stimulation with phytohaemagglutinin (PHA). Using the toxin α-amanitin it has been possible to demonstrate that within 4 h of the addition of PHA there is a two-fold increase in the amanitin-resistant polymerase activity (polymerase A) with little increase in the sensitive polymerase activity (polymerase B). 24 h following PHA stimulation the amanitin-resistant activity is stimulated 4–5 fold and the amanitin-sensitive activity less than two-fold. The susceptibility of this increased amanitin-resistant activity to low doses of actinomycin D both in vivo and in vitro indicates that the amanitin-resistant enzyme is mainly engaged in ribosomal RNA precursor synthesis. These changes in DNA-dependent RNA polymerase activity closely correspond to the observed changes in ribosomal and non-ribosomal RNA synthesis following lymphocyte stimulation.The increased polymerase A activity is diminished by a 1 h incubation of the cells with cycloheximide added 24 h after PHA whereas polymerase B activity remains unaffected. This indicates that the polymerase A activity observed after transformation is dependent on continuing protein synthesis.In our incubation conditions the polymerase activity observed in isolated nuclei appeared to be almost wholly attributable to elongation of nascent RNA molecules attached to the endogenous DNA template.