Mediator Influences Schizosaccharomyces pombe RNA Polymerase II-dependent Transcription in Vitro

Abstract

The fission yeast Schizosaccharomyces pombe has proved an important model system for cross-species comparative studies of many fundamental processes in the eukaryotic cell, such as cell cycle control and DNA replication. The RNA polymerase II transcription machinery is, however, still relatively poorly understood in S. pombe, partially due to the absence of a reconstituted in vitro transcription system. We have now purified S. pombe RNA polymerase II and its general initiation factors TFIIB, TFIIF, TFIIE, and TFIIH to near homogeneity. These factors enable RNA polymerase II to initiate transcription from the S. pombe alcohol dehydrogenase promoter (adh1p) when combined with Saccharomyces cerevisiae TATA-binding protein. We use our reconstituted system to examine effects of Mediator on basal transcription in vitro. S. pombe Mediator exists in two distinct forms, a free form, which contains the spSrb8, spTrap240, spSrb10, and spSrb11 subunits, and a smaller form, which lacks these four subunits and associates with RNA polymerase II to form a holoenzyme. We find that spSrb8/spTrap240/spSrb10/spSrb11 containing Mediator repress basal transcription, whereas Mediator lacking these subunits has a stimulatory effect on transcription. Our findings thus demonstrate that the spSrb8/spTrap240/spSrb10/spSrb11 subcomplex governs the ability of Mediator to stimulate or repress basal transcription in vitro.

Mcs2 and a novel CAK subunit Pmh1 associate with Skp1 in fission yeast

“…2C). This suggests that the Pmh1 immunoprecipitation could contain other fission yeast TFIIH subunits [38]. These observations together with the demonstration that T-loop phosphorylation of Mcs6 is not essential [6] are consistent with a model where association of Pmh1 with the Mcs6 CDK and Mcs2 cyclin alleviates the requirement of T-loop phosphorylation as observed in other species [8,9,21,42], and that Pmh1 represents the Mat1 homolog in fission yeast.…”

“…The sequence of the primers is available upon request. The Pmh1-TAP strain has been described elsewhere [38]. The Mcs2-3HA strain was a kind gift from Beach [1].…”

The cyclin-dependent kinase 8 module sterically blocks Mediator interactions with RNA polymerase II

“…In addition, a subgroup of Srb proteins, Med12͞Srb8, Med13͞Srb9, Cdk8 (cyclin-dependent kinase 8)͞Srb10, and CycC͞Srb11, forms a specific module (the Cdk8 module) that is variably present in Mediator preparations (10,11). The smaller, core Mediator (S Mediator) lacking the Cdk8 module has a stimulatory effect on basal transcription in vitro (5,12). The larger form of Mediator (L Mediator), containing the Cdk8 module, instead represses basal transcription in vitro, and genetic analysis also indicates that the Cdk8 module is involved in the negative regulation of genes in vivo (13).…”

Cdk8 Is Essential for Preimplantation Mouse Development

“…In contrast to the core Mediator involved in transcriptional activation, the Cdk8 submodule is needed for transcriptional repression in vitro in both yeast and mammalian systems (33,39,41,42). The Cdk8 submodule consists of Cdk8 and cyclin C (24,26,44), together with Med12/Srb8 and Med13/Srb9 (3,4,37), and the loss of any of these four subunits results in a similar upregulation of a subset of genes in budding yeast but has little effect on the viability of cells (17,49).…”