The Mediator complex

What is it? The Mediator complex is an enormous, multi-subunit protein complex that regulates gene expression in eukaryotic cells. It acts as a molecular circuit board that integrates physiological and developmental signals and transmits them to RNA polymerase II (Pol II), the enzyme charged with transcription of all protein-encoding genes. Although initially discovered as a cofactor required for activator-dependent transcription, Mediator also stimulates activator-independent (basal) transcription. Furthermore, Mediator can act as a co-repressor that facilitates transcriptional silencing. Mediator activity is not limited to its effects on transcription initiation, but it can also affect elongation and perhaps all stages of the transcription cycle.How was Mediator discovered? First, a little background information. The 12-subunit Pol II complex (Rpb1–Rpb12) is all that is required for its catalytic activity, i.e. DNA template-directed RNA synthesis. However, Pol II is unable to recognize promoter DNA on its own and instead requires five general transcription factors (GTFs) — TFIIB, TFIID, TFIIE, TFIIF and TFIIH. The GTFs and Pol II assemble in a defined order on promoter DNA to form a pre-initiation complex (PIC). The PIC is sufficient for accurate start-site selection, but does not respond to transcriptional activators. Indeed, as the GTFs were purified to homogeneity, the Pol II response to activators became increasingly weaker. This provided a biochemical assay that led to the discovery of Mediator as a component of a yeast cell extract that dramatically stimulated activator-dependent transcription. Accordingly, Mediator is a key link in the pathway leading from enhancer-bound activators to promoter-bound Pol II.Purification of Mediator revealed that several of its subunits had been identified previously in genetic screens for regulators of gene expression in yeast. In fact, a single genetic selection for suppressors of a growth defect associated with truncation of the carboxy-terminal domain (CTD) of the Rpb1 subunit of Pol II identified nine different Mediator components (SRB genes). Additional subunits had also been identified in other screens for mutants that either activated or repressed gene expression. Thus, the synergy of biochemistry and genetics led to the discovery of Mediator as an essential regulator of Pol II transcription. The identification of more than a third of its components as the products of SRB genes suggested that Mediator affects gene expression, at least in part, through the Pol II CTD. More on that in a moment...Is Mediator found in organisms other than yeast? Mediator appears to be a universal requirement for Pol II transcription. Several Mediator-like complexes were identified in transcription systems derived from fly, worm, mouse, rat and human cells. Each complex was named according to how it was discovered: TRAP (thyroid hormone receptor-associated proteins), SMCC (SRB/Mediator co-activator complex), ARC (activator-recruited complex), DRIP (vitamin D receptor-interacting proteins), CRSP (cofactor required for Sp1 activation) and PC2 (positive cofactor 2). In each case, these complexes were required for the response to transcriptional regulators in vitro. However, their relationship to each other and to Mediator was not immediately apparent due to subunit heterogeneity and to amino acid sequence divergence among orthologous subunits. Further characterization of these complexes established that all are metazoan counterparts of Mediator with a common modular structure that includes orthologs of all yeast subunits (Figure 1Figure 1). Metazoan Mediator complexes often include additional subunits not found in yeast Mediator and exist in multiple forms with variable subunit composition, reflecting more complex transcriptional regulatory programs in these organisms.Figure 1The Mediator complex.(A)The 25-subunit yeast Mediator complex consists of four structurally distinct modules: head, middle/arm, tail and kinase. Although a uniform nomenclature has been assigned to orthologous Mediator subunits from eukaryotic organisms, nine of the yeast subunits were identified in a genetic selection for suppressors of truncation of the Pol II carboxy-terminal domain (CTD); these genes were designated SRBs, including SRB8 to SRB11. (B) A ribbon model of the yeast Mediator head module, composed of 7 subunits, as seen from different angles. Med17 (cyan), Med11 (purple), Med22 (yellow), Med6 (green), Med8 (orange), Med18 (blue), and Med20 (red). This image was generously provided by Dr. Yuichiro Takagi (Indiana University School of Medicine).View Large Image | View Hi-Res Image | Download PowerPoint SlideIs Mediator required for expression of all genes? It seems so. Inactivation of the Med17 (SRB4 gene) subunit of yeast Mediator has the same deleterious effect on transcription as inactivation of the Rpb1 subunit of Pol II — essentially abolishing mRNA synthesis. Furthermore, a technique called chromatin immunoprecipitation (ChIP), which queries the association of specific proteins at any site in the genome, detected Mediator at the promoters of nearly all genes. As mentioned above, Mediator also stimulates activator-independent transcription. Indeed, in vitro transcription is so inefficient in the absence of Mediator that Mediator might be more appropriately viewed as a GTF, essential for all Pol II transcription.What does Mediator look like? Yeast Mediator consists of 25 subunits with a molecular mass of ∼1 million Daltons. Electron microscopy defined a modular structure consisting of four domains: head, middle/arm, tail and kinase (Figure 1Figure 1). The head module can stimulate activator-independent transcription, but does not respond to activators. Conversely, the tail module is the direct target of specific regulatory proteins and appears to be responsible for recruitment of Mediator to DNA. The middle/arm module is a highly flexible structure that orchestrates extensive conformational changes upon Mediator binding to Pol II.The hallmark of the kinase module is the Cdk8–cyclin C pair (Srb10–Srb11), which assembles with Med12 and Med13 (Figure 1Figure 1). All four of these subunits were discovered as SRB genes, implicating the kinase module in CTD function. This interaction might account, in part, for the co-repressor function of Mediator: Pol II is recruited to the promoter in hypo-phosphorylated form such that Cdk8-catalyzed CTD phosphorylation prevents Pol II assembly into the PIC. In human cells, a very different function for the Cdk8–cyclin C module has been reported: epigenetic silencing of neuronal genes via covalent histone modification. Furthermore, stimulatory effects on transcription have also been demonstrated for Cdk8 in yeast and metazoan cells, underscoring the enigmatic nature of the kinase module.What does the structure of Mediator reveal about its function? Just as high-resolution images of Pol II and the ribosome are unraveling the mechanisms of transcription and protein synthesis, structural studies of Mediator are providing insight into how this complex machine regulates transcription. The architecture of the head module was recently solved to high resolution, defining three structural domains — the neck, fixed jaw and movable jaw. These domains are built around a bundle of ten α-helices derived from five different subunits (Figure 1Figure 1). Structural and biochemical information suggests that the head module positions the Pol II CTD in proximity to TFIIH, where the TFIIH kinase (Cdk7) stimulates CTD phosphorylation, in this case (as opposed to Cdk8; see above) promoting dissociation of Pol II from Mediator, coinciding with the transition from initiation to elongation. These structures also provide an informative framework for interpreting the earlier SRB and other MED mutations, including secondary mutations that suppress primary mutations. For example, a Med17 defect is suppressed by a Med6 mutation, apparently by creating a stabilizing salt-bridge between the two subunits.Recent cryo-EM images of the head module in association with a minimal, functional PIC have also illuminated the mechanism of promoter binding. The jaws bind the Rpb4–Rpb7 subunits of Pol II, along with the ‘clamp’ that closes over the active site cleft. In the Pol II closed conformation, the cleft is large enough to accommodate singlestranded DNA, but not double-stranded DNA. Either the two strands of promoter DNA must melt prior to PIC assembly, or Pol II must undergo a conformational change that opens the cleft. The cryo-EM map suggests the latter: head interactions with Pol II cause repositioning of Rpb4–Rpb7 and a large rearrangement of the clamp, thereby opening the active site cleft. This model could account for the stimulatory effect of Mediator on basal transcription and suggests that clamp opening is a fundamental aspect of Mediator function.Does Mediator play a role in human disease? Mediator appears to play a role in diverse pathological states, including tumorigenesis, cardiovascular disease, neurological disease, and metabolic disorders. For instance, Med19 is upregulated in a variety of cancers, such as bladder cancer, breast cancer, lung tumors, and osteosarcoma. In addition, the X-linked Lujan and Opitz-Kaveggia mental retardation syndromes are caused by mutations in Med12, while mutations in Med13 have been found in patients with congenital heart defects. The fact that Mediator is a core component of the gene expression machinery indicates that defects in the transcriptional program are at the heart of these disorders.