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 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 . Prior studies identified a set of more than twenty DNA binding transcription factors bound at upstream pro- distinct polypeptides that compose the 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; 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 The initiation of messenger RNA synthesis is a major site for including the products of the Srb2, Srb4, Srb5, Srb6, and the regulation of expression. The initiation of eukaryotic Gal11 . 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 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 , 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, 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 [21]. cultured cells stably expressing a FLAG-tagged Med subunit. MudPIT is an exquisitely sensitive ‘‘shotgun proteomics’’ ap- proach for identifying the constituents of complex mixtures 3. Subunit composition of the mammalian Med complex of proteins [42,43]. In MudPIT, all of the proteins in a mixture are digested without prior separation into peptides, which are Multiprotein mammalian Med-like complexes have been iso- then separated by two-dimensional cation exchange and re- lated in several laboratories by a variety of procedures and verse phase HPLC and analyzed by in-line tandem mass include the thyroid hormone receptor-associated proteins/ spectrometry. SRB-Med containing cofactor (TRAP/SMCC) [22–25], activa- MudPIT analyses of multiprotein complexes purified from tor-recruited factor-large (ARC-L) [26,27], vitamin D recep- HeLa cell lines stably expressing either FLAG-MED10, tor-interacting proteins (DRIP) [28], positive cofactor 2 FLAG-MED9, FLAG-MED29, FLAG-MED19, FLAG- (PC2) [25], cofactor required for Sp1 activation (CRSP) MED28, FLAG-MED26 [41], or FLAG-MED8 (unpublished [29,27], mouse Med [30], and rat Med [31–34] complexes. Clon- data) identified 37 proteins present in all seven preparations ing and analysis of the genes encoding subunits of these com- but not in the control. Among those proteins were 29 of 30 plexes revealed that many bear striking sequence similarity to proteins previously identified as components of various mam- yeast Med subunits. These include obvious orthologs of S. malian Med complexes characterized in different laboratories cerevisiae Med subunits Srb7, Med6, Med7, Nut2, Srb10, (Fig. 1). The 30th protein, cyclin C, was identified by MudPIT and Srb11. In addition, bioinformatic and biochemical analy- in all but one preparation, but its presence was confirmed in all ses identified among the subunits of mammalian Med-like by Western blotting. Taken together, these results argue that complexes potential orthologs of all S. cerevisiae Med subunits all of the proteins previously identified as components of the except for Med2, Pgd1/Hrs1, and Nut1 [32–36]. Interestingly, various mammalian Med-like complexes are indeed bona fide although the mammalian MED1 protein is predicted to be Med subunits. an ortholog of S. cerevisiae Med subunit Med1, it also contains The only additional proteins present in all seven Med prep- LXXLL motifs, which mediate ligand-dependent interaction arations but not in the control were subunits of RNA polymer- of mammalian Med with class I and class II nuclear receptors ase II, a MED13-related protein, MED13L, encoded by the not found in yeast. The MED1 protein was initially identified KIAA1025 open reading frame (ORF), and a cyclin-dependent 906 J.W. Conaway et al. / FEBS Letters 579 (2005) 904–908

From Literature Large Small MudPIT

Mammalian S. cerevisiae ratMED TRAP/SMCC ARC DRIP mMED PC2 CRSP HeLa Control F:Med10 F:Med9 F:Med29 F:Med19 F:Med28 F:Med8 F:Med26 Med1 Med1 Med2 Med2 Pgd1/Hrs1 Med3 Med4 Med4 Nut1 Med5 Med6 Med6 Med7 Med7 Med8 Med8 Cse2 Med9 Nut2 Med10 Med11 Med11 Srb8 Med12 Srb9 Med13 Med13L Rgr1 Med14 Gal11 Med15 Sin4 Med16 Srb4 Med17 Srb5 Med18 Rox3 Med19 Srb2 Med20 Srb7 Med21 Srb6 Med22 Med23 Med24 Med25 Med26 Med27 Med28 Med29 Med30 Soh1 Med31 Srb10 Cdk8 Srb10 Cdk11 Srb11 cyclin C * *

Fig. 1. Comparison of mammalian Med subunits identified in different laboratories and by MudPIT. Mammalian Med subunits identified in the rat Med, TRAP/SMCC, ARC, DRIP, mouse Med (mMED), PC2, CRSP, and MudPIT analyses are indicated in blue. Proteins not detected are indicated in yellow. The asterisk in cyclin C MudPIT data from F:Med9 and F:Med26 expressing HeLa cells indicates that cyclin C was not detected in these Med preparations by MudPIT, but was detected by Western blotting. kinase, Cdk11 (Accession No. NP_055891), which is closely re- 5. Conclusions and future prospects lated to the Med subunit Cdk8. Mutations in MED13L are associated with a congenital heart defect called transposition The definition of a set of consensus mammalian Med sub- of the great arteries (TGA), suggesting that MED13 and units provides a solid foundation for future studies systemati- MED13L have non-redundant functions and may regulate cally dissecting the functions of individual subunits in the expression of distinct populations of genes. MudPIT anal- reconstitution of the mammalian Med and its associated activ- yses of multiprotein complexes purified from HeLa cell lines ities. Among the major questions to be addressed is how the stably expressing either FLAG-Cdk8 or FLAG-Cdk11 has re- Med promotes communication between DNA binding regula- vealed the existence of distinct forms of Med that contain tory proteins and RNA polymerase II and the general initia- either Cdk8 or Cdk11 (unpublished data). Finally, a tion factors. It is now well-established that the Med from MED12-related protein, MED12L, encoded by the TRAL- yeast, Drosophila, and yeast is capable of binding directly to PUSH ORF (Accession No. NP_443728) was identified by the transcriptional activation domains of many DNA binding MudPIT analysis of a mammalian Med complex isolated from regulatory proteins, which appear to function in part to recruit HEK 293 cells but not from HeLa cells (unpublished data), Med to genes [44–46]. A variety of evidences accumulated to suggesting that MED12L may be a component of a cell-type date supports the intriguing model that different DNA binding or tissue-specific form of Med. regulatory proteins may target Med to genes through direct J.W. Conaway et al. / FEBS Letters 579 (2005) 904–908 907 interactions with different Med subunits. Although the details [16] Xiao, Z., McGrew, J.T., Schroeder, A.J. and Fitzgerald-Hayes, of interactions between mammalian Med subunits and tran- M. (1993) Mol. Cell. Biol. 13, 4691–4702. scriptional activation domains are still emerging, documented [17] Hengartner, C.J., Thompson, C.M., Zhang, J., Chao, D.M., Liao, S.M., Koleske, A.J., Okamura, S. and Young, R.A. (1995) Genes examples include the interactions of MED1, MED23, and Dev. 9, 897–910. MED25 with nuclear receptors, VP16, and E1A and Elk1, [18] Koh, S.S., Ansari, A.Z., Ptashne, M. and Young, R.A. (1998) respectively [46–49]. Thus, future studies illuminating the Mol. Cell 1, 895–904. mechanisms underlying targeting of Med by the myriad [19] Asturias, F.J., Jiang, Y.W., Myers, L.C., Gustafsson, C.M. and Kornberg, R.D. (1999) Science 283, 985–987. DNA binding regulatory proteins active in a given cell will [20] Davis, J.A., Takagi, Y., Kornberg, R.D. and Asturias, F.J. (2002) be critical for an in-depth understanding of Med function Mol. Cell 10, 409–415. and gene transcription. [21] Lorch, Y., Beve, J., Gustafsson, C.M. and Kornberg, R.D. (2000) In addition, in light of evidence that Med is likely to be pres- Mol. Cell 6, 197–201. ent in cells in multiple, functionally distinct forms, a major aim [22] Fondell, J.D., Guermah, M., Malik, S. and Roeder, R.G. (1999) Proc. Natl. Acad. Sci. USA 96, 1959–1964. of future studies will be to identify the repertoire of Med [23] Gu, W., Malik, S., Ito, M., Yuan, C.X., Fondell, J.D., Zhang, X., assemblies and subassemblies and to elucidate their functions. Martinez, E., Qin, J. and Roeder, R.G. (1999) Mol. Cell 3, 97–108. Studies to date have identified distinct Med forms including [24] Ito, M., Yuan, C.X., Malik, S., Gu, W., Fondell, J.D., Yamam- and lacking the Cdk8/cyclin C/MED12/MED13 module and ura, S., Fu, Z.Y., Zhang, X., Qin, J. and Roeder, R.G. (1999) Mol. Cell 3, 361–370. have obtained tantalizing evidence that Med including the [25] Malik, S., Gu, W., Wu, W., Qin, J. and Roeder, R.G. (2000) Mol. Cdk8 module may lack associated RNA polymerase II and Cell 5, 753–760. can function as a transcriptional repressor under some condi- [26] Naar, A.M., Beaurang, P.A., Zhou, S., Abraham, S., Solomon, tions ([27,47,50,51] and our unpublished results). With the W. and Tjian, R. (1999) Nature 398, 828–832. identification of Med forms containing either Cdk8 or Cdk11 [27] Taatjes, D.J., Naar, A.M., andel, F., Nogales, E. and Tjian, R. (2002) Science 295, 1058–1062. and combinations of MED12/MED13 and the related [28] Rachez, C., Lemon, B.D., Suldan, Z., Bromleigh, V., Gamble, M., MED12L/MED13L proteins, the number of predicted Med Naar, A.M., Erdjument-Bromage, H., Tempst, P. and Freedman, forms that may perform non-redundant functions in cells has L.P. (1999) Nature 398, 824–828. increased further. Thus, future studies cataloguing the number [29] Ryu, S., Zhou, S., Ladurner, A.G. and Tjian, R. (1999) Nature 397, 446–450. of distinct Med forms and establishing the mechanisms of their [30] Jiang, Y., Veschambre, P., Erdjument-Bromage, H., Tempst, P., interconversions and their likely gene-specific functions will be Conaway, J.W., Conaway, R.C. and Kornberg, R.D. (1998) Proc. essential. Natl. Acad. Sci. USA 95, 8538–8543. [31] Brower, C.S., Sato, S., Tomomori-Sato, C., Kamura, T., Pause, A., Stearman, R., Klausner, R.D., Malik, S., Lane, W.S., Acknowledgement: This work was supported in part by National Insti- Sorokina, I., Roeder, R.G., Conaway, J.W. and Conaway, R.C. tutes of Health Grant R37 GM041628. (2002) Proc. Natl. Acad. Sci. USA 99, 10353–10358. [32] Sato, S., Tomomori-Sato, C., Banks, C.A., Parmely, T.J., Sorokina, I., Brower, C.S., Conaway, R.C., and Conaway, J.W. (2003) J. Biol. Chem. Published on-line October 22, 2003; 10.1074/ References jbc.C300444200. [33] Sato, S., Tomomori-Sato, C., Banks, C.A., Sorokina, I., Parmely, [1] Kim, Y.J., Bjorklund, S., Li, Y., Sayre, M.H. and Kornberg, R.D. T.J., Kong, S.E., Jin, J., Cai, Y., Lane, W.S., Brower, C.S., (1994) Cell 77, 599–608. Conaway, J.W. and Conaway, R.C. (2003) J. Biol. Chem. 278, [2] Nonet, M.L. and Young, R.A. (1989) Genetics 123, 715–724. 15123–15127. [3] Thompson, C.M., Koleske, A.J., Chao, D.M. and Young, R.A. [34] Tomomori-Sato, C., Sato, S., Parmely, T.J., Banks, C.A., (1993) Cell 73, 1361–1375. Sorokina, I., Florens, L., Zybailov, B., Washburn, M.P., Brower, [4] Koleske, A.J. and Young, R.A. (1994) Nature 368, 466–469. C.S., Conaway, R.C. and Conaway, J.W. (2003) J. Biol. Chem. [5] Suzuki, Y., Nogi, Y., Abe, A. and Fukasawa, T. (1988) Mol. Cell. 279, 5846–5851. Biol. 8, 4991–4999. [35] Boube, M., Joulia, L., Cribbs, D.L. and Bourbon, H.-M. (2002) [6] Myers, L.C., Gustafsson, C.M., Bushnell, D.A., Lui, M., Erdj- Cell 110, 143–151. ument-Bromage, H., Tempst, P. and Kornberg, R.D. (1998) [36] Bourbon, H.M., Aguilera, A., Ansari, A.Z., Asturias, F.J., Genes Dev. 12, 45–54. Berk, A.J., Bjorklund, S., Blackwell, T.K., Borggrefe, T., Carey, [7] Li, Y., Bjorklund, S., Jiang, Y.W., Kim, Y.J., Lane, W.S., M., Carlson, M., Conaway, J.W., Conaway, R.C., Emmons, Stillman, D.J. and Kornberg, R.D. (1995) Proc. Natl. Acad. Sci. S.W., Fondell, J.D., Freedman, L.P., Fukasawa, T., Gutafsson, USA 92, 10864–10868. C.M., Han, M., He, X., Herman, P.K., Hinnebusch, A.G., [8] Gustafsson, C.M., Myers, L.C., Li, Y., Redd, M.J., Lui, M., Holmber, S., Holstege, F.C., Jaehning, J.A., Kim, Y.J., Kuras, Erdjument-Bromage, H., Tempst, P. and Kornberg, R.D. (1997) L., Leutz, A., Lis, J.T., Meisterernest, M., Naar, A.M., J. Biol. Chem. 272, 48–50. Nasmyth, K., Parvin, J.D., Ptashne, M., Reinberg, D., Ronne, [9] Gustafsson, C.M., Myers, L.C., Beve, J., Spahr, H., Lui, M., H., Sadowski, I., Sakurai, H., Sipiczki, M., Sternberg, P.W., Erdjument-Bromage, H., Tempst, P. and Kornberg, R.D. (1998) Stillman, D.J., Strich, R., Struhl, K., Svejstrup, J.Q., Tuck, S., J. Biol. Chem. 273, 30851–30854. Winston, F., Roeder, R.G. and Kornberg, R.D. (2004) Mol. [10] Sakai, A., Shimizu, Y. and Hishinuma, F. (1988) Genetics 119, Cell, 553–557. 499–506. [37] Lee, J.W., Choi, H.S., Gyuris, J., Brent, R. and Moore, D.D. [11] Jiang, Y.W. and Stillman, D.J. (1992) Mol. Cell. Biol. 12, 4503– (1995) Mol. Endocrinol. 9, 243–254. 4514. [38] Zhu, Y., Qi, C., Jain, S., Rao, M.S. and Reddy, J.K. (1997) J. [12] Chen, S., West, R.W., Johnson, S.L., Gans, H., Kruger, B. and Biol. Chem. 272, 25500–25506. Ma, J. (1993) Mol. Cell. Biol. 13, 831–840. [39] Ito, M., Yuan, C.-X., Okano, H.J., Darnell, R.B. and Roeder, [13] Rosenblum-Vos, L.S., Rhodes, L., Evangelista, C.C., Boayke, R.G. (2000) Mol. Cell 5, 683–693. K.A. and Zitomer, R.S. (1991) Mol. Cell. Biol. 11, 5639–5647. [40] Baek, H.J., Malik, S., Qin, J. and Roeder, R.G. (2002) Mol. Cell. [14] Santos-Rosa, H., Clever, B., Heyer, H.D. and Aguilera, A. (1996) Biol. 22, 2842–2852. Genetics 142, 705–716. [41] Sato, S., Tomomori-Sato, C., Parmely, T.J., Florens, L., Zybailov, [15] Tabtiang, R.K. and Herskowitz, I. (1998) Mol. Cell. Biol. 18, B., Swanson, S.K., Banks, C.A., Jin, J., Cai, Y., Washburn, M.P., 4707–4718. Conaway, J.W. and Conaway, R.C. (2004) Mol. Cell 14, 685–691. 908 J.W. Conaway et al. / FEBS Letters 579 (2005) 904–908

[42] Wolters, D., Washburn, M.P. and Yates, J.R. (2001) Anal. Chem. [47] Mittler, G., Stu¨hler, T., Santolin, L., Kremmer, E., Lottspeich, F., 73, 5683–5690. Berti, L. and Meisterernst, M. (2003) EMBO J. 22, 6494–6504. [43] Washburn, M.P., Wolters, D. and Yates III, J.R. (2001) Nat. [48] Yang, F., DeBeaumont, R., Zhou, S. and Naar, A.M. (2004) Biotechnol. 19, 242–247. Proc. Natl. Acad. Sci. USA 101, 2339–2344. [44] Myers, L.C. and Kornberg, R.D. (2000) Annu. Rev. Biochem. 69, [49] Stevens, J.L., Cantin, G.T., Wang, G., Shevchenko, A., Shev- 729–749. chenko, A. and Berk, A.J. (2002) Science 296, 755–758. [45] Kim, T.W., Kwon, Y.J., Kim, J.M., Song, Y.H., Kim, S.N. [50] Samuelsen, C.O., Baraznenok, V., Khorosjutina, O., Spa˚hr, H., and Kim, Y.J. (2004) Proc. Natl. Acad. Sci. USA 101, 12153– Kieselbach, T., Holmberg, S. and Gustafsson, C.M. (2003) Proc. 12158. Natl. Acad. Sci. USA 100, 6422–6427. [46] Malik, S. and Roeder, R.G. (2000) Trends Biochem. Sci. 25 (6), [51] Mo, X., Kowenz-Leutz, E., Xu, H. and Leutz, A. (2004) Mol. Cell 277–283. 13, 241–250.