electron microscopy ͉ transcription ͉ protein structure ͉ yeast ͉ Schizosaccharomyces pombe T he Mediator complex acts as an interface between genespecific regulatory proteins and the basal RNA polymerase II (pol II) transcription machinery (1). Mediator functions as a key regulator of pol II-dependent genes in Saccharomyces cerevisiae (2), and depletion of human Mediator from nuclear extracts abolishes transcription by pol II (3). The C-terminal domain of pol II (CTD) has an important role for the Mediator function (4, 5), and no fewer than nine SRB genes, encoding for Mediator subunits, were originally identified in a screen for mutants that suppress the cold-sensitive phenotype of a CTD truncation mutant (6). In S. cerevisiae and Schizosaccharomyces pombe, the Mediator complex interacts directly with the unphosphorylated CTD and forms a holoenzyme (5). Based on shape analysis of the low-resolution projection maps, the S. cerevisiae Mediator structure has been divided into three compact and visually distinguishable modules: head, middle, and tail domains of approximately equal mass (7).The subunit composition of S. cerevisiae Mediator has been studied in detail, and 21 proteins are bona fide members of the core Mediator complex (1,8,9). 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).The Cdk8 module influences pol II interactions with Mediator, and only S Mediator can interact with pol II and form a holoenzyme complex (11). The molecular mechanism by which the Cdk8 module negatively regulates pol II interactions has not been clarified, but it has been hypothesized that the negative effect of the Cdk8 module on eukaryotic transcription is caused by Cdk8-dependent phosphorylation of CTD. The hyperphosphorylated form of CTD would bind less tightly to Mediator, which may result in dissociation of pol II from the holoenzyme complex (14, 15).Here, we use the S. pombe system to investigate the molecular basis for the distinct functional properties of S and L Mediator. We find that the Cdk8 module binds to the pol II-binding cleft of Mediator, where it sterically blocks interactions with the polymerase. In contrast to earlier assumptions, the Cdk8 kinase activity is dispensable for negative regulation of pol II interactions with Mediator. It should be noted that the structure and function of Mediator appears conserved in fungi and metazoan cells, and to simplify comparisons with other experimental systems, throughout this study we use the recently proposed unifying Mediator nomenclature ...

In Saccharomyces cerevisiae Mediator, a subgroup of proteins (Srb8, Srb9, Srb10, and Srb11) form a module, which is involved in negative regulation of transcription. Homologues of Srb10 and Srb11 are found in some mammalian Mediator preparations, whereas no clear homologues have been reported for Srb8 and Srb9. Here, we identify a TRAP240͞ARC250 homologue in Schizosaccharomyces pombe and demonstrate that this protein, spTrap240, is stably associated with a larger form of Mediator, which also contains conserved homologues of Srb8, Srb10, and Srb11. We find that spTrap240 and Sch. pombe Srb8 (spSrb8) regulate the same distinct subset of genes and have indistinguishable phenotypic characteristics. Importantly, Mediator containing the spSrb8͞spTrap240͞spSrb10͞spSrb11 subunits is isolated only in free form, devoid of RNA polymerase II. In contrast, Mediator lacking this module associates with the polymerase. Our findings provide experimental evidence for recent suggestions that TRAP230͞ARC240 and TRAP240͞ARC250 may indeed be the Srb8 and Srb9 homologues of mammalian Mediator. Apparently Srb8͞ TRAP230͞ARC240, Srb9͞TRAP240͞ARC250, Srb10, and Srb11 constitute a conserved Mediator submodule, which is involved in negative regulation of transcription in all eukaryotes.Schizosaccharomyces pombe ͉ RNA polymerase II ͉ transcription ͉ repression

Mediator is an evolutionary conserved coregulator complex required for transcription of almost all RNA polymerase II-dependent genes. The Schizosaccharomyces pombe Mediator consists of two dissociable components—a core complex organized into a head and middle domain as well as the Cdk8 regulatory subcomplex. In this work we describe a functional characterization of the S. pombe Mediator. We report the identification of the S. pombe Med20 head subunit and the isolation of ts alleles of the core head subunit encoding med17+. Biochemical analysis of med8ts, med17ts, Δmed18, Δmed20 and Δmed27 alleles revealed a stepwise head domain molecular architecture. Phenotypical analysis of Cdk8 and head module alleles including expression profiling classified the Mediator mutant alleles into one of two groups. Cdk8 module mutants flocculate due to overexpression of adhesive cell-surface proteins. Head domain-associated mutants display a hyphal growth phenotype due to defective expression of factors required for cell separation regulated by transcription factor Ace2. Comparison with Saccharomyces cerevisiae Mediator expression data reveals that these functionally distinct modules are conserved between S. pombe and S. cerevisiae.

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.

With the identification of eight new polypeptides, we here complete the subunit characterization of the Schizosaccharomyces pombe RNA polymerase II holoenzyme. The complex contains homologs to all 10 essential gene products present in the Saccharomyces cerevisiae Mediator, but lacks clear homologs to any of the 10 S. cerevisiae components encoded by nonessential genes. S. pombe Mediator instead contains three unique components (Pmc2, -3, and -6), which lack homologs in other cell types. Presently, pmc2 ϩ and pmc3 ϩ have been shown to be nonessential genes. The data suggest that S. pombe and S. cerevisiae share an essential protein module, which associates with nonessential speciesspecific subunits. In support of this view, sequence analysis of the conserved yeast Mediator components Med4 and Med8 reveals sequence homology to the metazoan Mediator components Trap36 and Arc32. Therefore, 8 of 10 essential genes conserved between S. pombe and S. cerevisiae also have a metazoan homolog, indicating that an evolutionary conserved Mediator core is present in all eukaryotic cells. Our data suggest a closer functional relationship between yeast and metazoan Mediator than previously anticipated.RNA polymerase II ͉ transcription