View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector FEBS Letters 579 (2005) 904–908 FEBS 29061 Minireview The mammalian Mediator complex Joan Weliky Conawaya,b,c,*, Laurence Florensa, Shigeo Satoa, Chieri Tomomori-Satoa, Tari J. Parmelya, Tingting Yaoa, Selene K. Swansona, Charles A.S. Banksa, Michael P. Washburna, Ronald C. Conawaya,b a Stowers Institute for Medical Research, Kansas City, MO 64110, USA b Department of Biochemistry and Molecular Biology, Kansas University Medical Center, Kansas City, KS 66160, USA c Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73190, USA Received 25 October 2004; accepted 2 November 2004 Available online 24 November 2004 Edited by Gunnar von Heijne and Anders Liljas expressed in eukaryotes from yeast to man, is composed of Abstract The multiprotein Mediator (Med) complex is an evo- lutionarily conserved transcriptional regulator that plays impor- more than twenty subunits and has been named Mediator tant roles in activation and repression of RNA polymerase II (Med) for its role in mediating transcriptional signals from transcription. Prior studies identified a set of more than twenty DNA binding transcription factors bound at upstream pro- distinct polypeptides that compose the Saccharomyces cerevisiae moter elements and enhancers to RNA polymerase II and Mediator. Here we discuss efforts to characterize the subunit the general initiation factors bound at the core promoter sur- composition and associated activities of the mammalian Med rounding the transcriptional start site. complex. Ó 2004 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. 2. Saccharomyces cerevisiae Mediator Keywords: Mass spectrometry; Mediator; Messenger RNA synthesis; MudPIT; RNA polymerase II; Transcription factor The Mediator was identified and first purified to near homo- geneity from S. cerevisiae by Kornberg and coworkers, by vir- tue of its ability to promote activator-dependent transcription by purified RNA polymerase II and the general initiation fac- tors in vitro [1]. This brought to light, for the first time, the 1. Introduction enormous complexity of the S. cerevisiae Med complex, by demonstrating that it was composed of some 20 proteins The initiation of messenger RNA synthesis is a major site for including the products of the Srb2, Srb4, Srb5, Srb6, and the regulation of gene expression. The initiation of eukaryotic Gal11 genes. The S. cerevisiae Srb genes were initially isolated messenger RNA synthesis is an elaborate biochemical process in a genetic screen for extragenic suppressors of an RNA poly- that is catalyzed by the 12 subunit enzyme RNA polymerase II merase II mutant containing a partial deletion of the heptad and requires, for simply a basal level of initiation, a minimum repeats in the carboxyl terminal domain (CTD) of its largest of five general initiation factors designated TFIIB, TFIID, subunit [2] and found to copurify in a high molecular mass TFIIE, TFIIF, and TFIIH, which comprise more than 20 dis- complex that was capable of supporting activator-dependent tinct polypeptides. Regulation of transcription initiation by RNA polymerase II transcription in vitro [3,4]. The Gal11 gene RNA polymerase II is further complicated by the requirement was initially isolated as gene required for transcription of for a very large, multisubunit ‘‘adaptor’’ that bridges RNA galactose-inducible genes [5]. polymerase II and its myriad DNA binding regulatory proteins Further characterization of the purified S. cerevisiae Med and transduces both positive and negative signals that turn on complex by Kornberg and coworkers led to the identification and off messenger RNA synthesis in response to the ever as Med subunits of the product of the Srb7 gene [6], the prod- changing microenvironment of the cell. This adaptor, which ucts of the Rgr1 and Sin4 genes [7], the product of the Rox3 is now known to be evolutionarily conserved and ubiquitously gene [8], the product of the Pgd1 Hrs1 gene [6], a collection of previously uncharacterized S. cerevisiae proteins, which they designated Med1, Med2, Med4, Med6, Med7, and Med8 [6], *Corresponding author. Fax: 816 926 2091. the products of the Cse2, Nut1, and Nut2 genes [9], and a pre- E-mail address: [email protected] (J.W. Conaway). viously uncharacterized protein, which they designated Med11 Abbreviations: ARC, activator-recruited cofactor; CRSP, cofactor re- [9]. Yeast strains carrying Rgr1 and Sin4 mutations exhibit quired for Sp1 activation; Cse2, chromosome segregation 2; CTD, similar transcriptional defects. Yeast strains carrying an Rgr1 carboxyl terminal domain; DRIP, vitamin D receptor-interacting pr- mutation failed to repress transcription of the Suc2 gene in otein; Med, Mediator; MudPIT, multidimensional protein identifica- the presence of glucose [10]. Yeast strains carrying a Sin4 tion technology; Nut, negative regulation of URS2; ORF, open reading frame; Rgr1, resistance to glucose repression 1; SMCC, Srb- mutation are defective in repressing transcription of the Gal1 Med-containing cofactor; Srb, suppressor of RNA polymerase B; and HO endonuclease genes [11,12]. Yeast strains carrying TRAP, thyroid hormone receptor-associated protein Rox3 mutations fail to repress transcription of the heme-regu- 0014-5793/$30.00 Ó 2004 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2004.11.031 J.W. Conaway et al. / FEBS Letters 579 (2005) 904–908 905 lated Cyc7 gene [13]. The Pdg1/Hrs1 gene was isolated in a ge- as TRIP2 (thyroid receptor interacting protein 2) and PBP netic screen for extragenic suppressors of the hyperrecombina- (PPAR binding protein) in yeast two-hybrid screens, by its tion phenotype of yeast carrying a deletion of the Hpr1 gene ability to interact in a ligand-dependent manner with the thy- [14]. Yeast strains carrying Pdg1/Hrs1 mutations were found roid hormone and PPAR receptors, respectively [37,38].In to be defective in activation of transcription of the Gal10 gene light of evidence that the MED1 protein is not required for [14]. The Nut1 and Nut2 genes were initially isolated in a genet- structural integrity of the mammalian Med complex and thus ic screen for yeast mutants defective in transcriptional activa- far appears to mediate activation of RNA polymerase II tran- tion by the DNA binding activator Swi4 [15]. The Cse2 gene scription only by nuclear receptors, the MED1 protein may was isolated in a genetic screen for yeast strains defective in play a specialized role in transcriptional regulation in mamma- chromosome segregation [16]. lian cells [39]. In addition to defining the subunit composition of the Despite significant similarities in the subunit compositions S. cerevisiae Med complex, Kornberg and coworkersÕ charac- of mammalian Med complexes isolated in different laborato- terization of the biochemical properties of their purified Med ries, apparent differences were notable (Fig. 1). The MED30 preparations led to the initial identification of many of the and MED31 proteins were initially identified only in the known Med-associated activities [1]. They and others pro- TRAP/SMCC complex [23,40]; the MED25 protein only in vided direct evidence that Med binds tightly not only to the the ARC, DRIP, and CRSP complexes [26–29]; the transcriptional activation domains of DNA binding transcrip- MED26 protein only in the CRSP and DRIP complexes tion factors [17,18], but also to RNA polymerase II to form a [27–29]; the MED8 protein only in the ARC and rat Med ‘‘holoenzyme’’ [1,4], consistent with the model that Med par- complexes [26]; the MED18 protein only in the mouse and ticipates directly in activator-dependent recruitment of poly- rat Med complexes [30,32]; and the MED9, MED11, merase to promoters. Although it is not yet completely MED19, MED22, MED28, and MED29 only in rat Med clear as to how Med interacts with RNA polymerase II, evi- [32–34]. dence suggests that yeast Med can bind directly to the heptad repeats in enzymeÕs CTD [1], and, through analysis of elec- tron micrographs of the holoenzyme, it appears that Med 4. Proteomic analysis of the mammalian Med complex makes multiple contacts with polymerase, with important contacts provided by Med interactions with the Rpb3 and Although the precise explanation for the apparent differ- Rpb11 polymerase subunits [19,20]. Perhaps not surprising ences in subunit compositions of the various mammalian in light of MedÕs significant interaction with RNA polymer- Med complexes characterized in different laboratories is cur- ase II in the preinitiation complex, yeast Med exerts potent rently not known, recent proteomic analyses suggests that stimulation of transcription initiation in reactions carried most of the Med-associated proteins identified in different lab- out in the absence of activators [1]. Binding of Med to oratories are bona fide subunits of the complex [41]. In these RNA polymerase II was found to stimulate phosphorylation experiments, multidimensional protein identification technol- of the CTD heptad repeats by the TFIIH-associated CTD ki- ogy (MudPIT) was used to characterize the subunit composi- nase [1]. Finally, S. cerevisiae Med was found to have associ- tion of a variety of mammalian Med complexes purified by ated histone acetyltransferase (HAT) activity likely carried anti-FLAG agarose immunoaffinity chromatography from out by its Nut1 subunit