UBC Theses and Dissertations
Studies on brain nuclear RNA polymerase and chromatin transcription Singh, Vijendra Kumar
In order to elaborate the nature of the mechanisms controlling the transcription of genes in the cells of higher organisms, studies have been carried out on brain nuclear RNA polymerase and the transcription of brain chromatin. Foremost, suitable conditions were developed for the solubilization of RNA polymerase in high yields from purified nuclei of beef brain. The solubilized enzyme was partially purified by ammonium sulphate fractionation followed by DEAE-cellulose chromatography. By this procedure, two DNA-dependent RNA polymerase activities, designated as RNA polymerase I and RNA polymerase II, were resolved. These were partly characterized on the basis of their differing catalytic properties. RNA polymerase II exhibits a preferential requirement for Mn⁺⁺ as the divalent cation and heat-denatured DNA as the template, is markedly stimulated by 0.2 M KCl and selectively inhibited by the toxin, α-amanitin. On the other hand, polymerase I prefers Mg⁺⁺ as the divalent cation and native DNA as the template, is considerably inhibited by 0.2 M KC1 and is not affected by α -amanitin. The capacity of RNA synthesis in vitro by RNA polymerase isolated from brain nuclei was markedly enhanced by polyamines such as spermidine or spermine. Spermidine exerted a much more pronounced effect on polymerase II than on polymerase I. Evidence is presented suggesting that RNA polymerase II activity may be preferentially stimulated by spermidine. Yeast RNA inhibited the activity of polymerase II and spermidine counteracted this inhibition almost completely, indicating that spermidine may act by circumventing the product-inhibition. The product of the polymerase II reaction sedimented at around 18 S in a sucrose-density gradient and appeared to be a complex of the type Enzyme-DNA-RNA. The template activity of the isolated brain chromatin for brain nuclear RNA polymerase II and E. coli RNA polymerase was less than 25% than that of the pure calf thymus DNA. This greatly repressed template capacity of the chromatin was probably due to the acid-soluble chromosomal proteins. The brain polymerase II was 3-4 times more active with acid-treated chromatin than pure DNA as template whereas the E. coli enzyme was almost equally active with either of these two templates. The RNA synthesised on either native or acid-treated chromatin as template by brain polymerase II was somewhat smaller in size than the RNA made on pure DNA as template. It appears that the control of transcription of genes in mammalian cells could be mediated by the multiplicity of the transcriptive enzyme, RNA polymerase, and by the physiological state of the template as well.
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