tailieunhanh - Báo cáo khoa học: Mediator is required for activated transcription in a Schizosaccharomyces pombe in vitro system

RNA polymerase II (RNAPII) requires a set of general transcription factors )TFIIA, TFIIB, TFIID, TFIIE, TFIIF and TFIIH)to initiate transcription from a gene promoterin vitro. General transcription factors have been isolated from Saccharomyces cerevisiae,rat, human and Drosophila,and their corresponding cDNAs have been cloned. In this report, we describe a reconstitutedin vitro transcription system that consists of the following prepara-tions of factors from the yeastSchizosaccharomyces pombe: affinity-purified RNAPII, TFIIH, and recombinant TBP, TFIIB, TFIIE and TFIIF. . | Eur. J. Biochem. 271 2561-2572 2004 FEBS 2004 doi Mediator is required for activated transcription in a Schizosaccharomyces pombe in vitro system Evelyn Tamayo Giuliano Bernal Ursula Teno and Edio Maldonado Programa de Biologia Celular y Molecular Facultad de Medicina Instituto de Ciencias Biomedicas Universidad de Chile Santiago Chile RNA polymerase II RNAPII requires a set of general transcription factors - TFIIA tFiIB TFIID TfIIE TFIIF and TFIIH - to initiate transcription from a gene promoter in vitro. General transcription factors have been isolated from Saccharomyces cerevisiae rat human and Drosophila and their corresponding cDNAs have been cloned. In this report we describe a reconstituted in vitro transcription system that consists of the following preparations of factors from the yeast Schizosaccharomyces pombe affinity-purified RNAPII TFIIH and recombinant TBP TFIIB TFIIE and TFIIF. We show that this system can support basal transcription from the adenovirus major late promoter when purified RNAPII is used and activated transcription when the RNAPII holoenzyme RNAPII plus the Mediator proteins is included in the reaction. In contrast the TATA binding protein-associated factors had no effect on transcriptional activation in our Sc. pombe system. These results indicate that Sc. pombe uses the same set of general transcription factors as other eukaryotes and that the Mediator is involved in activated transcription. The ability to achieve basal levels of transcription from protein-encoding genes in eukaryotes requires RNA polymerase II RNAPII and a set of additional proteins called general transcription factors GTFs . The GTFs have been purified to homogeneity from HeLa cells rat liver Drosophila and the yeast Saccharomyces cerevisiae and have been named tFiIA TFIID tFiIE TFIIF TFIIB and TFIIH 11 . Tie cDNAs that encode these factors have been isolated and their amino acid sequences show a high degree of evolutionary .