Article | Published:

Reconstitution of active human core Mediator complex reveals a critical role of the MED14 subunit

Nature Structural & Molecular Biology volume 21, pages 10281034 (2014) | Download Citation

Abstract

The evolutionarily conserved Mediator complex is a critical coactivator for RNA polymerase II (Pol II)-mediated transcription. Here we report the reconstitution of a functional 15-subunit human core Mediator complex and its characterization by functional assays and chemical cross-linking coupled to MS (CX-MS). Whereas the reconstituted head and middle modules can stably associate, basal and coactivator functions are acquired only after incorporation of MED14 into the bimodular complex. This results from a dramatically enhanced ability of MED14-containing complexes to associate with Pol II. Altogether, our analyses identify MED14 as both an architectural and a functional backbone of the Mediator complex. We further establish a conditional requirement for metazoan-specific MED26 that becomes evident in the presence of heterologous nuclear factors. This general approach paves the way for systematic dissection of the multiple layers of functionality associated with the Mediator complex.

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Acknowledgements

We thank T. Richmond (Institute of Molecular Biology and Biophysics, Eidgenössische Technische Hochschule Zurich) for the MultiBac baculovirus system, J. Fernandez-Martinez and M.P. Rout (Rockefeller University, Laboratory of Cellular and Structural Biology) for assistance with the offline Agilent HPLC system and M. Guermah (Rockefeller University, Laboratory of Biochemistry and Molecular Biology) for discussion. Funding for this work was provided by US Department of Defense grant W81XWH-13-1-0172 (R.G.R.) and by US National Institute of Health grants CA129325 (R.G.R.), GM090929 (R.G.R. and S.M.), GM103511 (B.T.C.), GM109824 (B.T.C.) and GM103314 (B.T.C.). M.A.C. was supported by an American Cancer Society Eastern Division–New York Cancer Research Fund Postdoctoral Fellowship.

Author information

Affiliations

  1. Laboratory of Biochemistry and Molecular Biology, Rockefeller University, New York, New York, USA.

    • Murat A Cevher
    • , Dan Li
    • , Sohail Malik
    •  & Robert G Roeder
  2. Laboratory of Mass Spectrometry and Gaseous Ion Chemistry, Rockefeller University, New York, New York, USA.

    • Yi Shi
    •  & Brian T Chait

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Contributions

M.A.C., S.M., R.G.R., Y.S. and B.T.C. designed the experiments and wrote the manuscript. M.A.C. carried out biochemical experiments including cDNA preparations, reconstitutions, in vitro transcriptions and coimmunoprecipitation experiments. D.L. helped M.A.C. in the generation of partial head-module complexes (in Supplementary Fig. 5d). Y.S. carried out the CX-MS experiments.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Robert G Roeder.

Integrated supplementary information

Supplementary information

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  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–6

  2. 2.

    Supplementary Table 1

    DSS cross-link dataset of the reconstituted Mediator complex

  3. 3.

    Supplementary Table 2

    Annotated HCD MS/MS spectra of the cross-linked peptides identified from the reconstituted Mediator complex

  4. 4.

    Supplementary Data Set 1

    Uncropped gels from Figs. 1 and 2

  5. 5.

    Supplementary Data Set 2

    Uncropped gels from Figs. 3 and 4

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https://doi.org/10.1038/nsmb.2914

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