Molecular architecture of the yeast Mediator complex

Abstract

The 21-subunit Mediator complex transduces regulatory information from enhancers to promoters, and performs an essential role in the initiation of transcription in all eukaryotes. Structural information on two-thirds of the complex has been limited to coarse subunit mapping onto 2-D images from electron micrographs. We have performed chemical cross-linking and mass spectrometry, and combined the results with information from X-ray crystallography, homology modeling, and cryo-electron microscopy by an integrative modeling approach to determine a 3-D model of the entire Mediator complex. The approach is validated by the use of X-ray crystal structures as internal controls and by consistency with previous results from electron microscopy and yeast two-hybrid screens. The model shows the locations and orientations of all Mediator subunits, as well as subunit interfaces and some secondary structural elements. Segments of 20–40 amino acid residues are placed with an average precision of 20 Å. The model reveals roles of individual subunits in the organization of the complex.DOI: http://dx.doi.org/10.7554/eLife.08719.001

Integrative structure and functional anatomy of a nuclear pore complex

“…The Cryo-ET density restraint corresponded to the cross-correlation between the Gaussian Mixture Model (GMM) representation of most Nups and the GMM representation of the Cryo-ET density map 103,122–124 (Fig. 3; Extended Data Figs.…”

Evidence for Multiple Mediator Complexes in Yeast Independently Recruited by Activated Heat Shock Factor

“…Most models of yeast Mediator have suggested that it is a monolithic, 21-subunit complex that reversibly associates with the CKM (16,20,21,49). Studies using conventional mutants have identified the existence of subcomplexes (generally comprised of tail and scaffold subunits) whose existence has been inferred using both in vivo and cell-free assays (49)(50)(51).…”

“…The flexibility and extended shape of the head permit interactions with Pol II at three putative interaction surfaces-on Rpb1 (within its C-terminal domain [CTD]), Rpb3, and the Rpb4/Rpb7 subcomplex-as well as with other components of the transcription initiation complex, including TBP, TFIIB and TFIIH (13)(14)(15)(16)(17)(18). In both yeast and humans, Med17 serves as the structural hub within the head module, as well as a major link between head and middle through its interaction with the scaffold subunit Med14 and the middle subunits Med7, Med10, and Med21 (19)(20)(21).…”

“…For example, the conserved subunit Med15 serves as the target of the yeast activators Gcn4, Gal4, Oaf1, Pdr1/Pdr3, and Hsf1 (10,(23)(24)(25)(26)(27), as well as mammalian sterol regulatory elementbinding protein (SREBP) (11). Med14, generally assigned to either the tail or middle, has recently been shown to serve as a central scaffold necessary for integrating the three separate modules of the core into a single functional entity (16,(19)(20)(21).…”

Functional interplay between Mediator and TFIIB in preinitiation complex assembly in relation to promoter architecture

“…The spatial organization of the Mediator modules was revised recently (Tsai et al 2014;Wang et al 2014). Med14 links the middle and tail module and, according to a recent model of entire yeast Mediator, makes extensive contacts with subunits from all three core modules (Robinson et al 2015). A reconstitution of the active human core Mediator complex revealed a critical role of the Med14 subunit in Mediator architecture and function (Cevher et al 2014).…”