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 [1]. The cDNAs that encode these factors have been isolated, and their amino acid sequences show a high degree of evolutionary conservation. These findings indicate that the transcriptional machinery is highly conserved among eukaryotes.An in vitro transcription assay that consists of purified RNAPII and recombinant GTFs can carry out basal transcription but cannot respond to gene-specific transcriptional activators. Activated transcription requires additional multiprotein complexes named coactivators. The main coactivators required for activated transcription in in vitro systems are the TFIID complex and the Mediator [2]. Recent work suggests that the TFIID complex, which contains the TATA binding protein (TBP) and other TBPassociated factors (TAFs), plays an important role in facilitating activation by gene-specific transcription factors as wells as in recognition of the TATA box and other core promoter sequences (necessary for both basal and activated transcription) [3]. Mediator is a large multiprotein complex that is brought to promoters by DNA-bound, gene-specific transcriptional regulatory proteins and helps these proteins to communicate with factors bound to the core promoter. Mediator is required for transcription in vivo and for optimal levels of both basal and activated transcription in vitro in nuclear extracts from human cells [4,5]. Components of both the TFIID complex and Mediator are conserved from yeast to humans.The yeast Schizosaccharomyces pombe can be genetically manipulated and has served as an exce...