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 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.