The protein composition of human Mediator-P.5 and Mediator-P.85 complexes is described in Wu et al. (2003). This table is adapted from Bourbon et al. (2004). t Subunits detected in S. cerevisiae, but not yet identified in C. elegans, D. melanogaster, or H. sapiens Mediator complexes. § Subunits also detected in D. melanogaster, but not yet identified in C. elegans or H. sapiens Mediator complexes.

The protein composition of human Mediator-P.5 and Mediator-P.85 complexes is described in Wu et al. (2003). This table is adapted from Bourbon et al. (2004). t Subunits detected in S. cerevisiae, but not yet identified in C. elegans, D. melanogaster, or H. sapiens Mediator complexes. § Subunits also detected in D. melanogaster, but not yet identified in C. elegans or H. sapiens Mediator complexes.

MED 12-MED13-CDK8-CycC module not found in the smaller Mediator complexes, such as Mediator-P.85, CRSP and PC2 (see Table 4.2). In contrast, the small Mediator complex has a unique polypeptide, MED26/ CRSP70, normally absent in the large complex. The rest of Mediator components seem to be commonly shared between large and small Mediator complexes, although the identities of some subunits remain to be characterized. The structures of yeast and murine Mediator as well as human TRAP, ARC and CRSP complexes have been resolved by electron microscopy at 30-40Ä (Asturias et al., 1999; Dotson et al., 2000; Taatjes et al., 2002). Comparison of these structures reveals a similarity in the overall organization of Mediator complexes. In general, three visible domains (named head, middle and tail in yeast Mediator) that may adapt to distinct conformations when complexed with activators or with the CTD are clearly distinguishable.

B: Head Module

The head module of Mediator, consisting of MED6, MED8, MED11, MED 17, MED 18, MED 19, MED20, and MED22, forms the base of a roughly triangle-shaped Mediator complex. This triangular complex undergoes a drastic conformational change upon association with pol II, resulting in an arc-shaped structure in which the head module at the leading edge serves as a major docking site for pol II. This structural information is consistent with yeast genetic screens showing direct interaction between pol II and MED17/Srb4, MED18/Srb5, MED20/Srb2, and MED22/Srb6 respectively (Thompson et al., 1993). Moreover, MED 17 and MED 18 also interact with transcriptional activators p53 and Gal4-VP16, respectively (Ito et al., 1999; Lee et al., 1999), suggesting that activator-induced conformational changes (Taatjes et al., 2002) may further enhance head module-mediated recruitment of pol II.

C: Middle and Tail Modules

The middle module of Mediator contains MEDI, MED4, MED5, MED7, MED9, MEDIO, MED21, and MED31, whereas the tail module includes MED2, MED3, MED 14, MEDI5, and MED 16 (Boube et al., 2002; Guglielmi et al., 2004). From the structures of yeast Mediator, it is obvious that, besides head module-pol II interaction, additional contacts are made between the Rpbl, Rpb3, Rpb6, and Rpbll subunits of pol II with regions of Mediator extending from the head module to the intersection between middle and tail modules (Davis et al., 2002). Clearly, these extensive contacts may help Mediator unfold from its compact triangular shape, in which the middle and tail modules are not clearly visible in the absence of pol II, to a more extended conformation when bound to pol II. Despite the extensive contacts, the DNA-binding cleft and interaction surfaces for other components of the general transcription machinery are still accessible on pol II (Davis et al., 2002). Since MEDI in the middle module can interact with multiple nuclear receptors (Yuan et al., 1998), MED 14 and MED 15 in the tail module can associate with Gal4-VP16 (Lee et al., 1999; Park et al., 2000), it is likely that conformational changes induced upon activator binding to the middle and, especially the tail, module further contribute to activator-facilitated recruitment of pol II by Mediator (Taatjes et al., 2002; Wu et al., 2003).

D: MED12-MED13-CDK8-CycC Module

A dissociable module, which contains MED12/Srb8, MED 13/Srb9, CDK8/Srbl0, and CycC/Srbll normally found in the large, but not small, Mediator complex (Borggrefe et al., 2002; Samuelsen et al., 2003), seems to contact mainly the middle module, via CDK8 interaction with MEDI and MED4 (Kang et al., 2001), and also the head module, through MED 13 interaction with MED 17 (Guglielmi et al., 2004). This CDK8 module, which could be isolated as a free entity, is able to phosphorylate serines 2 and 5 of the pol II CTD

(Borggrefe et al., 2002). That transcription could be inhibited by CDK8-mediated phosphorylation of the CTD occurring prior to PIC assembly (Hengartner et al., 1998) or through phosphorylation on serines 5 and 304 of the cyclin H subunit of TFIIH by recombinant CDK8-CycC pair or by the NAT complex (Akoulitchev et al., 2000) suggests that the CDK8 module may function as a repression module in the context of Mediator. This view is supported by biochemical evidence showing that the large form of ARC (ARC-L) is transcriptionally inactive (Taatjes et al., 2002) and the coactivating activity of Mediator-P.5 was slightly enhanced when CDK8-CycC was immunodepleted from the large Mediator complex (Wu et al., 2003). Clearly, removal of CDK8 in yeast by nutrient deprivation (Holstege et al., 1998) or in mouse P19 embryonic carcinoma cells following all-trans retinoic acid (tRA) treatment (Pavri et al., 2005) enhances transcription from a subset of cellular genes normally suppressed by CDK8. In the latter case, it was further demonstrated that dissociation of CDK8 following tRA treatment converts Mediator from a transcriptionally suppressed state to an activated complex at the tRA-targeted RAR(32 gene promoter, indicating that CDK8 indeed functions in the context of a repression module in vivo.

E: Functional Properties of Mediator

In addition to the inhibitory activity conferred by the CDK8 repression module, Mediator is an authentic coactivator able to stimulate both basal and activator-dependent transcription (Kim et al., 1994). This stimulating activity of Mediator clearly relies on its ability to serve as a bridging molecule in transducing activation signals typically from activator-tail module to head module-pol II. Although Mediator can be isolated via its direct interactions with activators or with the CTD, direct biochemical evidence demonstrating that Mediator indeed functions by enhancing activator-facilitated entry of pol II to the PIC has only become possible after all the general transcription factors, cofactors, and pol II are available in purified forms devoid of any contaminating activities (Wu et al., 2003). From this in Wiro-reconstituted transcription system, we learn that the large form of Mediator complexes, such as Mediator-P5, has intrinsic coactivating activity able to stimulate activator-dependent transcription, whereas the small form of Mediator complexes, such as Mediator-P.85, only enhances basal transcription. Interestingly, the coactivator function of Mediator can occur in the absence of TFIID TAFs, suggesting that Mediator and TAFs may play some redundant roles in the transcriptional process. Indeed, it has been shown that TAFs can also enhance pol II entry to the PIC in the presence of transcriptional activators (Wu and Chiang, 2001). Besides targeting on pol II, Mediator has the ability to enhance TBP binding to the TATA box. This TATA-enhancing activity may help stabilize the promoter-bound scaffold complex, which contains TFIIA, TFIID, TFIIE, TFIIH, and Mediator (Yudkovsky et al., 2000), to facilitate reinitiation of transcription from the same promoter.

As defined in TFIID, Mediator exhibits multiple enzymatic activities. The kinase activity of Mediator, inherent to the CDK8 subunit, can phosphorylate the CTD of pol II (Hengartner et al., 1998; Sun et al., 1998; Borggrefe et al., 2002), the cyclin H subunit of TFIIH (Akoulitchev et al., 2000), and general cofactor PC4 (Gu et al., 1999). In addition, Mediator has been reported to exhibit HAT activity, residing in the MED5/Nutl subunit, that preferentially acetylates histones H3 and H4 in the context of both free histones and chromatin (Lorch et al., 2000). However, no HAT activity has been reported in Mediator complexes purified from other species to date. Interestingly, some components of Mediator, such as MED8, are able to assemble into an E3 ubiquitin ligase involved in proteasome-mediated degradation (Brower et al., 2002), suggesting that Mediator may have ubiquitin ligase activity yet to be uncovered. These enzymatic activities clearly account for some roles of Mediator in the transcriptional process, including a possible stimulation of TFIIH kinase activity on the CTD (Kim et al., 1994). Many of these interesting questions remain to be addressed.

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