We used single-particle electron microscopy to characterize the structure and subunit organization of the Mediator Head module that controls Mediator-RNA polymerase II (RNAPII) and Mediatorpromoter interactions. The Head module adopts several conformations differing in the position of a movable jaw formed by the Med18-Med20 subcomplex. We also characterized, by structural, biochemical and genetic means, the interactions of the Head module with TATA-binding protein (TBP) and RNAPII subunits Rpb4 and Rpb7. TBP binds near the Med18-Med20 attachment point and stabilizes an open conformation of the Head module. Rpb4 and Rpb7 bind between the Head jaws, establishing contacts essential for yeast-cell viability. These results, and consideration of the structure of the Mediator-RNAPII holoenzyme, shed light on the stabilization of the pre-initiation complex by Mediator and suggest how Mediator might influence initiation by modulating polymerase conformation and interaction with promoter DNA.Transcriptional regulation is focused on the initiation process, which entails recruitment of RNAPII and the general transcription factors to a promoter. Both basal and activated transcription are critically dependent on the Mediator complex [1][2][3][4][5] , which conveys regulatory signals to RNAPII. Consistent with its essential role, the Mediator complex is conserved in sequence and structure throughout the eukaryotes [6][7][8] . Unfortunately, despite the paramount importance of Mediator, the mechanism of action of the complex remains unclear, highlighting the significance of investigating its structure, subunit organization and conformational variability.Biochemical and structural analyses have shown that Mediator has a modular organization. Biochemically defined subunit modules appear to correspond to structural modules identified by structural studies. Recent cryo-electron microscopy (EM) analysis of Mediator (
AUTHOR CONTRIBUTIONST.I., F.C. and Y.T. expressed, purified and biochemically characterized recombinant Head module and Head module subcomplexes and provided recombinant Rpb4 and Rpb7 and TBP; Y.T. and T.I. designed and carried out Head-Rpb4-Rpb7 and Head-TBP binding assays; K.Y. and Y.T. designed and carried out assays to test genetic interaction of Mediator subunits with Rpb4; Y.T. carried out in vitro transcription assays; G.C. carried out all EM data collection and analysis; G.C., Y.T. and F.J.A. discussed and interpreted all results; F.J.A. supervised EM structural analysis and wrote the manuscript in collaboration with G.C. and Y.T.
COMPETING INTERESTS STATEMENTThe authors declare no competing financial interests.Reprints and permissions information is available online at http://npg.nature.com/reprintsandpermissions/. Of all Mediator modules, the Head is perhaps the most critical, as evidenced by cessation of mRNA synthesis at nearly all promoters in vivo when Head module function is compromised in a Med17 temperature-sensitive Saccharomyces cerevisiae mutant strain [9][10][11] . Consistent with this obse...
SUMMARY
Mediator, the multi-subunit complex that plays an essential role in the regulation of transcription initiation in all eukaryotes, was isolated using an affinity purification protocol that yields pure material suitable for structural analysis. Conformational sorting of yeast Mediator single particle images characterized the inherent flexibility of the complex and made possible calculation of a cryo-EM reconstruction. Comparison of free and RNA polymerase II (RNAPII)-associated yeast Mediator reconstructions demonstrates that intrinsic flexibility allows structural modules to reorganize and establish a complex network of contacts with RNAPII. We demonstrate that, despite very low sequence homology, the structures of human and yeast Mediators are surprisingly similar and the structural rearrangement that enables interaction of yeast Mediator with RNAPII parallels the structural rearrangement triggered by interaction of human Mediator with a nuclear receptor. This suggests that the topology and structural dynamics of Mediator constitute important elements of a conserved regulation mechanism.
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