Analysis of
            <i>Schizosaccharomyces pombe</i>
            Mediator reveals a set of essential subunits conserved between yeast and metazoan cells

Proc. Natl. Acad. Sci. U.S.A.

Lindahl

et al. 2006

Self Cite

198

168

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 ...

Section: Electron Microscopy Of the S Pombe Mediator And Pol II Holomentioning

confidence: 76%

“…Immunoblot analysis for Med17 and Med18 was performed as described in ref. 21. Recombinant S. pombe Cdk8 (amino acids 2-293) fused to GST was overproduced in Escherichia coli BL21 (DE3) pLysS cells (Stratagene, La Jolla, CA) and purified from inclusion bodies as described in ref.…”

Section: Methodsmentioning

confidence: 99%

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

Elmlund

Proc. Natl. Acad. Sci. U.S.A.

Lindahl

et al. 2006

Self Cite

198

168

“…After washing with 20 ml of tobacco etch virus (TEV) protease cleavage buffer, we eluted the Mediator by incubation for 1 h at ϩ16°C with 200 units of TEV protease in 2 ml of TEV buffer. The identification of spSrb8, spTrap240, spSrb10, and spSrb11 by matrix-assisted laser desorption ioniza-tion͞time-of-flight (MALDI-TOF) MS analysis of in-gel digested proteins was described (19). SRB10͞11 Kinase Assay.…”

Section: Methodsmentioning

confidence: 99%

TRAP230/ARC240 and TRAP240/ARC250 Mediator subunits are functionally conserved through evolution

Samuelsen¹,

Proc. Natl. Acad. Sci. U.S.A.

Khorosjutina

et al. 2003

Self Cite

108

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

“…The identification of proteins in this report, by MALDI-TOF analysis of in-gel digested proteins was essentially as described previously (12).…”

Section: Tataaatatattagtatggaattcgagctcgtttaaac-3јmentioning

confidence: 99%

“…We have reported previously on the purification and characterization of the S. pombe Mediator (11)(12)(13). The Mediator complex is essential for basal and regulated expression of nearly all pol II-dependent genes in S. cerevisiae (14), and depletion of human Mediator from nuclear extracts abolishes transcription by pol II (15).…”

mentioning

confidence: 99%

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

Spåhr

Khorosjutina

Journal of Biological Chemistry

et al. 2003

Self Cite

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.