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Mediator Head module structure and functional interactions

Nature Structural & Molecular Biology volume 17, pages 273–279 (2010)Cite this article

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Abstract

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 Mediator-promoter 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.

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Figure 1: Mediator and Head module structure.
Figure 2: EM analysis of Head module and subcomplexes.
Figure 3: Head–TBP interactions.
Figure 4: Head module–Rpb4–Rpb7 interaction.
Figure 5: Interaction of the Head module with components of the mPIC and a possible mechanism for initiation regulation.

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Acknowledgements

We thank I. Berger (European Molecular Biology Laboratory France) for providing reagents and help to establish the MultiBac expression system and C. Kaplan (Texas A&M Univ.), G. Hartzog (Univ. of California, Santa Cruz) and M. Goebl (Indiana Univ.) for providing yeast vectors and advising on the implementation of genetic experiments. This work was supported by US National Institutes of Health grant R01 GM67167 (F.J.A.) and US National Science Foundation grant MCB 0843026 (Y.T.), an American Heart Association Scientist Development Award 0735395N (Y.T.) and a Human Frontier Science Program long-term fellowship (T.I.).

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Authors and Affiliations

  1. Department of Cell Biology, The Scripps Research Institute, La Jolla, California, USA

    Gang Cai & Francisco J Asturias

  2. Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, Indiana, USA

    Tsuyoshi Imasaki, Kentaro Yamada, Francesco Cardelli & Yuichiro Takagi

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  1. Gang Cai
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  2. Tsuyoshi Imasaki
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  6. Francisco J Asturias
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Contributions

T.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.

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Correspondence to Yuichiro Takagi or Francisco J Asturias.

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Supplementary Figures 1–5 and Supplementary Table 1 (PDF 352 kb)

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Cai, G., Imasaki, T., Yamada, K. et al. Mediator Head module structure and functional interactions. Nat Struct Mol Biol 17, 273–279 (2010). https://doi.org/10.1038/nsmb.1757

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