RESUMO
Templates were prepared with either the TATA box or transcription start sites of the yeast CYC1 promoter in a nucleosome. In both cases, initiation in an unfractionated yeast RNA polymerase II transcription system was abolished by the nucleosome. The inhibition appeared to be relieved by the activator protein Gal4-VP16 binding to a site upstream of the promoter. Inhibition was not relieved, however, in a transcription system reconstituted from purified components, indicating a requirement for additional factors for the effect of Gal4-VP16.
Assuntos
Cromatina/metabolismo , RNA Polimerase II/metabolismo , Moldes Genéticos , Transcrição Gênica , Sequência de Bases , DNA Fúngico , Proteínas Fúngicas/metabolismo , Dados de Sequência Molecular , Nucleossomos/metabolismo , Regiões Promotoras Genéticas , Saccharomyces cerevisiae/genética , TATA Box , Transativadores/metabolismoRESUMO
Fractionation of a yeast nuclear extract reveals at least four factors required in addition to RNA polymerase II for accurate initiation of transcription. One of these factors can be replaced by HeLa transcription factor IID or by its yeast counterpart expressed in Escherichia coli. Each of the remaining three factors can be replaced by a fraction from yeast whole cell extract, facilitating further purification of the factors.
Assuntos
RNA Polimerase II/metabolismo , Saccharomyces cerevisiae/enzimologia , Fatores de Transcrição/farmacologia , Transcrição Gênica , Núcleo Celular/enzimologia , Escherichia coli/metabolismo , Células HeLa/análise , Saccharomyces cerevisiae/ultraestrutura , Fator de Transcrição TFIIA , Fator de Transcrição TFIID , Fatores de Transcrição/isolamento & purificaçãoRESUMO
GRF2, an abundant yeast protein of Mr approximately 127,000, binds to the GAL upstream activating sequence (UASG) and creates a nucleosome-free region of approximately 230 bp. Purified GRF2 binds to sequences found in many other UASs, in the 35S rRNA enhancer, at centromeres, and at telomeres. Although GRF2 stimulates transcription only slightly on its own, it combines with a neighboring weak activator to give as much as a 170-fold enhancement. This effect of GRF2 is strongly distance-dependent, declining by 85% when 22 bp is interposed between the GRF2 and neighboring activator sites.
Assuntos
Cromatina/ultraestrutura , Proteínas de Ligação a DNA/metabolismo , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Sequências Reguladoras de Ácido Nucleico , Fatores de Transcrição , Leveduras/genética , Sequência de Bases , Cromatina/metabolismo , Cromatografia de Afinidade , Genes Fúngicos , Dados de Sequência Molecular , Transcrição GênicaRESUMO
Promoters were assembled in nucleosomes or ligated to nucleosomes and transcribed with SP6 RNA polymerase or with mammalian RNA polymerase II and accessory factors. Neither polymerase would initiate transcription at a promoter in a nucleosome, but once engaged in transcription, both polymerases were capable of reading through a nucleosome. In the course of readthrough transcription, the histones were displaced from the DNA, as shown by the exposure of restriction sites and by a shift of the template to the position of naked DNA in a gel. It may be true, in general, that processive enzymes will traverse regions of DNA organized in nucleosomes and displace histones.
