The transcription machinery in schizosaccharomyces pombe and its regulation

Abstract: The Mediator complex acts as a bridge, conveying regulatory information from enhancers and other control elements to the general transcription machinery. The Mediator was originally identified in Saccharomyces cerevisiae and is required for the basal and regulated expression of nearly all RNA polymerase II dependent genes. Mediator-like complexes have also been identified in higher eukaryotes and shown to play an essential role in transcription regulation. However, most of the subunits identified in these mammalian complexes displayed low or no significant sequence similarity with Mediator subunits previously identified in yeast. Our specific aim was to purify Mediator from Schizosaccharomyces pombe and to compare its subunit composition and function to S. cerevisiae and mammalian Mediators to shed light on the mechanism and evolution of Mediator dependent transcription regulation. In paper I and II, we purified the S. pombe Mediator in complex with RNA polymerase II. We showed that the S. pombe Mediator complex was considerably smaller than its S. cerevisiae counterpart containing only 13 subunits instead of 20. Three of the S. pombe subunits were species specific named PMC for Pombe Mediator Complex. Additionally, the S. pombe Mediator contained 10 subunits conserved in S. cerevisiae and 8 in metazoans. Genetics showed that the conserved subunits were essential for cell growth, whereas the species-specific subunits were non-essential. Our findings led us to propose that the Mediator consists of a set of core subunits conserved through evolution that is responsible for contacts with the general transcription machinery and a set of species-specific subunits that function as a dynamic interface for direct interactions with gene-specific activators. In paper III we analyzed the function of a specific Mediator subcomplex. Mediator from mammalian cells has been isolated in two different forms, the larger TRAP/Mediator complex and the smaller PC2/CRSP complex. The TRAP/Mediator complex contains 4 additional proteins, TRAP230, TRAP240, Srb10 and Srb11, which are absent in PC2/CRSP. We developed a purification scheme for the larger form of the S. pombe Mediator using the so-called tandem affinity purification tag (TAP). Our new purification procedure allowed to identify a novel form of Mediator, which also contained homologues to TRAP230, TRAP240, Srb10 and Srb11, which we denoted the TRAP240/Mediator. In paper IV we reconstituted a pure in vitro system for RNA polymerase II dependent transcription. We purified S. pombe general initiation factors TFIIB, TFIIF, TFIIE, and TFIIH to near homogeneity. These factors enabled highly purified RNA polymerase II to initiate transcription from the S. pombe alcohol dehydrogenase promoter (adh1p) when combined with S. cerevisiae TBP. We used the in vitro system to compare the activities of Mediator and the larger TRAP240/Mediator on basal transcription. We found that the smaller form of Mediator was able to stimulate transcription whereas the larger TRAP240/Mediator repressed transcription. Our studies lead us to propose a model for how the two forms of Mediator interact to regulate RNA polymerase II dependent transcription.