A temperature-sensitive mutation was obtained in Med6p, a component of the mediator complex from the yeast Saccharomyces cerevisiae. The mediator complex has been shown to enable transcriptional activation in vitro. This mutation in Med6p abolished activation of transcription from four of five inducible promoters tested in vivo. There was no effect, however, on uninduced transcription, transcription of constitutively expressed genes, or transcription by RNA polymerases I and III. Mediator-RNA polymerase II complex isolated from the mutant yeast strain was temperature sensitive for transcriptional activation in a reconstituted in vitro system due to a defect in initiation complex formation. A database search revealed the existence of MED6-related genes in humans and Caenorhabditis elegans, suggesting that the role of mediator in transcriptional activation is conserved throughout the evolution.Regulation of mRNA synthesis requires intermediary proteins that transduce regulatory signals from upstream transcriptional activator proteins to basal transcription machinery at the core promoter. A reconstituted in vitro transcription system composed only of basal transcription machinery (core-RNA polymerase II and pure general transcription factors) does not respond to upstream transcriptional activator proteins even though it is fully functional for basal transcription (26,36). Three types of intermediary factors that enable the basal transcription machinery to respond to transcriptional activator proteins bound to regulatory DNA sequences have been identified: (i) TAF II s, which associate with TATA-binding protein (TBP) to form TFIID (12, 13; for a review, see reference 53); (ii) mediator, which associates with RNA polymerase II to form a holo-polymerase (11,25,26,28; for a review, see reference 4); and (iii) coactivators such as human upstream stimulatory activity (USA) (36; for a review, see reference 23), mammalian CBP/P300 (for a review, see reference 19), yeast ADA complex (17), and HMG proteins (42,46). The interaction of these multiprotein complexes with activators and general transcription factors is essential for transcriptional regulation (3, 12-14, 18, 19, 43, 48, 57). In this paper, we focus on the functional analysis of one intermediary factor from Saccharomyces cerevisiae called mediator.The existence of mediator was originally suggested from a squelching assay, in which the addition of one activator interferes with stimulation of transcription by another activator, suggesting that the two activators compete for a common target (11,25). Squelching is specific for the activation domains of transcriptional activator proteins and could be relieved by addition of a partially purified yeast protein fraction but not by addition of known components of the basal transcription machinery. These results identified a novel activity termed mediator that is distinct from proteins required for basal transcription and that is essential for transcriptional activation. Fractionation of the yeast mediator activity yielded a...
Mediator proteins are required for transcriptional regulation of most genes in yeast. Mammalian Mediator homologs also function as transcriptional coactivators in vitro; however, their physiological role in gene-specific transcription is not yet known. To determine the role of Mediator proteins in the development of complex organisms, we purified putative Mediator complexes from Caenorhabditis elegans and analyzed their phenotypes in vivo. C. elegans Mediator homologs were assembled into two multiprotein complexes. RNA interference assays showed that the CeMed6, CeMed7, and CeMed10͞CeNut2 gene products are required for the expression of developmentally regulated genes, but are dispensable for expression of the ubiquitously expressed genes tested in this study. Therefore, the gene-specific function of Mediator as an integrator of transcriptional regulatory signals is evolutionarily conserved and is essential for C. elegans development.
The multisubunit Mediator complex of Saccharomyces cerevisiae is required for most RNA polymerase II (Pol II) transcription. The Mediator complex is composed of two subcomplexes, the Rgr1 and Srb4 subcomplexes, which appear to function in the reception of activator signals and the subsequent modulation of Pol II activity, respectively. In order to determine the precise composition of the Mediator complex and to explore the specific role of each Mediator protein, our goal was to identify all of the Mediator components. To this end, we cloned three previously unidentified Mediator subunits, Med9/Cse2, Med10/Nut2, and Med11, and isolated mutant forms of each of them to analyze their transcriptional defects. Differential display and Northern analyses of mRNAs from wild-type and Mediator mutant cells demonstrated an activator-specific requirement for each Mediator subunit. Med9/Cse2 and Med10/Nut2 were required, respectively, for Bas1/Bas2-and Gcn4-mediated transcription of amino acid biosynthetic genes. Gal11 was required for Gal4-and Rap1-mediated transcriptional activation. Med11 was also required specifically for MF␣1 transcription. On the other hand, Med6 was required for all of these transcriptional activation processes. These results suggest that distinct Mediator proteins in the Rgr1 subcomplex are required for activator-specific transcriptional activation and that the activation signals mediated by these Mediator proteins converge on Med6 (or the Srb4 subcomplex) to modulate Pol II activity.Regulation of mRNA synthesis by transcriptional activator proteins requires many diverse regulatory proteins collectively called transcriptional coactivators (for reviews, see references 2, 17, and 40). The TATA binding protein-associated factors (TAF II s), which compose the TFIID complex, and the multisubunit Mediator complex are the two major coactivators that enable the basal transcription machinery to respond to genespecific transcriptional regulatory proteins.TAF II s were initially identified in human and Drosophila as essential factors for transcriptional activation in a reconstituted transcription system (12, 33). Biochemical analysis of TFIID revealed a modular structure in which a large TAF II subunit, acting as a scaffold, binds to several distinct TAF II subunits, each of which interacts with specific transcriptional activator proteins (4). However, depletion or inactivation of TAF II s from the yeast Saccharomyces cerevisiae caused no obvious defect in transcriptional activation in vivo (28, 41). Therefore, it was proposed that TAF II s function as essential cofactors for transcription of only a subset of genes, rather than as general targets of transcriptional activators (1, 35).In contrast to the limited requirement for TAF II s, a second coactivator complex, the Mediator complex, appears to be required for the transcription of most RNA polymerase II (Pol II)-transcribed genes. The Mediator complex is required not only for transcriptional activation but also for the stimulation of basal transcription and h...
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