Med5 (Nut1) is identified here as a component of the Mediator tail region. Med5 is positioned peripherally to Med16 (Sin4) together with the three members of the putative Gal11 module, Med15 (Gal11), Med2, and Med3 (Pgd1). The biochemical analysis receives support from genetic interactions between med5⌬ and med15⌬ deletions. The med5⌬ and med16⌬ deletion strains share many phenotypes, including effects on mitochondrial function with enhanced growth on nonfermentable carbon sources, increased citrate synthase activity, and increased oxygen consumption. Deletion of the MED5 gene leads to increased transcription of nuclear genes encoding components of the oxidative phosphorylation machinery, whereas mitochondrial genes encoding components of the same machinery are down-regulated. We discuss a possible role for Med5 in coordinating nuclear and mitochondrial gene transcription.The multiprotein Mediator complex is required for basal and regulated expression of nearly all RNA polymerase II (pol II) 3 -dependent genes in the Saccharomyces cerevisiae genome. Mediator conveys regulatory information from enhancers and other control elements to the promoter (1). The functional activities identified for Mediator include stimulation of basal transcription, support of activated transcription, and enhancement of phosphorylation of the C-terminal domain of pol II by the transcription factor IIH kinase (2, 3). S. cerevisiae Mediator also contains a histone acetyltransferase (HAT) activity, which is not found in other eukaryotic Mediator complexes (4). The HAT activity was localized to Med5 (Nut1), a S. cerevisiae-specific protein, which lacks homologues in higher eukaryotes (4, 5). The MED5 gene was originally isolated in a screen for mutants that would suppress the Swi4/Swi6 dependence of a synthetic reporter gene containing part of the HO promoter (6). Several other genes encoding Mediator proteins were identified in the same screen, including MED10 (NUT2), MED16 (SIN4), MED19 (ROX3), MED12 (SRB8), MED13 (SRB9), CDK8 (SRB10), and CYCC (SRB11). The MED5 gene is nonessential in yeast. A deletion of MED5 relieves repression at the URS2 element in the HO promoter but only in combination with a mutant allele of either MED10 or CCR4 (6). These effects on the HO promoter were seen with a lacZ reporter gene but not at the endogenous HO gene locus. The in vivo role of Med5 in Mediator-dependent gene expression therefore remains an open question.In the presence of RNA pol II, Mediator adopts an extended conformation that embraces the globular pol II core complex (7). The extended structure reveals three distinct submodules of Mediator. Direct contacts are formed between pol II and the head and middle region (7,8). The largest part of Mediator is made up of an elongated tail region, which does not appear to contact pol II. Structural analysis of mutant Mediator complex has demonstrated that the tail region contains the Med2, Med3, Med15 (Gal11), and Med16, proteins, which are involved in interactions with a number of different activators,...

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 Mediator complex is required for the regulated transcription of nearly all RNA polymerase II-dependent genes. Here we demonstrate a new role for Mediator which appears to be separate from its function as a transcriptional coactivator. Mediator associates directly with heterochromatin at telomeres and influences the exact boundary between active and inactive chromatin. Loss of the Mediator Med5 subunit or mutations in Med7 cause a depletion of the complex from regions located near subtelomeric X elements, which leads to a change in the balance between the Sir2 and Sas2 proteins. These changes in turn result in increased levels of H4K16 acetylation near telomeres and in desilencing of subtelomeric genes. Increases in H4K16 acetylation have been observed at telomeres in aging cells. In agreement with this observation, we found that the loss of MED5 leads to shortening of the Saccharomyces cerevisiae (budding yeast) replicative life span.In Saccharomyces cerevisiae (budding yeast), telomeric DNA consists of imperfect tandem repeats of the consensus sequence (TG 1-3 ) n , with a combined length of about 300 nucleotides (21). Next to the telomere is the subtelomeric region, which often contains two types of repeats, the YЈ and X elements. The YЈ elements are between 4 and 12 kb long and are located next to the telomeres at many chromosome ends (26). The sizes of X elements vary, but they always contain a "core X" repeat region that is found at nearly all telomeres. Depending on how the YЈ and X elements are distributed, S. cerevisiae chromosome ends can be divided into X and X-YЈ types (22,23).Genes situated close to telomeres undergo reversible silencing, a phenomenon that has been termed the telomere position effect (TPE) (28). This effect was first observed when a reporter gene was inserted next to a telomeric TG 1-3 tract of an artificial telomere. TPE can also be observed at native yeast telomeres, but the phenomenon appears to be a bit more complex at these locations, since TPE varies between telomeres and in different strain backgrounds (29,40,41). The molecular basis of TPE is believed to be the Rap1, Ku, and Sir protein-mediated spreading of heterochromatin-like structures from the telomeric DNA inwards, which represses genes located in the subtelomeric region (42). According to this model, the Rap1/Ku/Sir structures are formed at telomeres and propagate toward the subtelomeres via interactions between the Sir proteins and histone tails. Sir2 is an active histone deacetylase that removes the acetyl group on lysine 16 of histone H4 (H4K16), which allows Sir3 and Sir4 to bind the nonacetylated histone tails (11, 35).As mentioned, TPE varies between individual chromosome ends, and the exact repeat structure of the subtelomeric region may in fact influence the spread of heterochromatin. YЈ elements counteract the spread of Sir proteins, and the YЈ regions display high levels of H4K16 acetylation. Furthermore, YЈ elements are highly enriched in nucleosomes and are transcriptionally active. In contrast, even X ...

The yeast Mediator complex is required for transcriptional regulation both in vivo and in vitro, and its function is conserved in all eukaryotes. Mediator interacts with both transcriptional activators and RNA polymerase II, but little is known about the mechanisms by which it operates at the molecular level. Here, we show that the cyclin-dependent kinase Srb10 interacts with, and phosphorylates, the Med2 subunit of Mediator both in vivo and in vitro. A point mutation of the single phosphorylation site in Med2 results in a strongly reduced expression of the REP1, REP2, FLP1, and RAF1 genes, which are all located on the endogenous 2-m plasmid. Combined with previous studies on the effects of SRB10͞SRB11 deletions, our data suggest that posttranslational modifications of Mediator subunits are important for regulation of gene expression.transcriptional regulation ͉ Srb11 ͉ RNA polymerase II T he Mediator complex was originally identified in Saccharomyces cerevisiae as an activity required for transcriptional activation in an in vitro transcription system reconstituted from highly purified RNA polymerase II (pol II) and general transcription factors (1, 2). Mediator was later purified to homogeneity and shown to be a complex composed of 20 subunits that interacts with the C-terminal domain of the largest pol II subunit (3, 4). More recent work has described several subunit-subunit interactions and subdomains within Mediator (5-8), and lowresolution structures of Mediator alone or in complex with pol II have been determined by three-dimensional reconstruction from electron micrographs of single particles (9, 10). These structures indicate a division of Mediator into distinct domains.In parallel with the identification of yeast Mediator, a pol II holoenzyme that comprised both Mediator subunits [a subset of general transcription factors and additional proteins (i.e., Srb8-11)] was purified (11). Nine SRB genes