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A conserved Mediator–CDK8 kinase module association regulates Mediator–RNA polymerase II interaction

Nature Structural & Molecular Biology volume 20, pages 611–619 (2013)Cite this article

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Abstract

The CDK8 kinase module (CKM) is a conserved, dissociable Mediator subcomplex whose component subunits were genetically linked to the RNA polymerase II (RNAPII) C-terminal domain (CTD) and individually recognized as transcriptional repressors before Mediator was identified as a pre-eminent complex in eukaryotic transcription regulation. We used macromolecular EM and biochemistry to investigate the subunit organization, structure and Mediator interaction of the Saccharomyces cerevisiae CKM. We found that interaction of the CKM with Mediator's middle module interferes with CTD-dependent RNAPII binding to a previously unknown middle-module CTD-binding site and with the holoenzyme formation process. Taken together, our results reveal the basis for CKM repression, clarify the origin of the connection between CKM subunits and the CTD and suggest that a combination of competitive interactions and conformational changes that facilitate holoenzyme formation underlie the mechanism of transcription regulation by Mediator.

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Figure 1: EM analysis and subunit organization of yeast CKM.
Figure 2: Mediator-CKM interaction in yeast.
Figure 3: Human Mediator-CKM interaction.
Figure 4: Mediator-CTD and Mediator-RNAPII interaction in yeast.
Figure 5: Interplay between CKM and RNAPII interaction with Mediator.
Figure 6: A model for CKM-dependent repression through obstruction of CTD-dependent Mediator-RNAPII interaction.

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Acknowledgements

This work was supported by US National Institutes of Health grants R01 67167 (F.J.A.) and RO1 GM41628 (R.C.C. and J.W.C.) and by a grant to the Stowers Institute from the Helen Nelson Medical Research Fund at the Greater Kansas City Community Foundation. Information about the human Mediator-CDK8 interaction came from reanalysis of samples originally provided by S. Malik and R. Roeder (Rockefeller University, New York, New York, USA). We thank Y. Takagi (Indiana University School of Medicine, Indianapolis, Indiana, USA) for providing a plasmid for GST-CTD expression, purified recombinant Gcn4 and head module and for helpful comments about the manuscript. We thank R.A. Young (Whitehead Institute for Biomedical Research, Cambridge, Massachusetts, USA) for providing yeast strains Z695 and Z735 bearing the Srb8-1 and Srb10-1 mutations. We also acknowledge the National Resource for Automated Macromolecular Microscopy (NRAMM).

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Authors and Affiliations

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

    Kuang-Lei Tsai & Francisco J Asturias

  2. Stowers Institute for Medical Research, Kansas City, Missouri, USA

    Shigeo Sato, Chieri Tomomori-Sato, Ronald C Conaway & Joan W Conaway

  3. Department of Biochemistry & Molecular Biology, Kansas University Medical Center, Kansas City, Kansas, USA

    Ronald C Conaway & Joan W Conaway

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  1. Kuang-Lei Tsai
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Contributions

All experiments, except those dealing with Mediator-CKM interaction in human cells, were designed by K.-L.T. and F.J.A. and carried out by K.-L.T. K.-L.T. and F.J.A. discussed and interpreted results and wrote the manuscript. Human Mediator-CKM interaction experiments (Fig. 3) were designed, discussed and interpreted by C.T.-S., S.S., R.C.C. and J.W.C. and carried out by C.T.-S. and S.S.

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Correspondence to Francisco J Asturias.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–6, Supplementary Table 1 and Supplementary Note (PDF 559 kb)

Supplementary Video 1

A movie illustrating the different orientations of Mediator-bound CKM. (MOV 2065 kb)

Supplementary Video 2

A movie illustrating the CTD-dependent interaction of RNAPII with Mediator. (MOV 1189 kb)

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Tsai, KL., Sato, S., Tomomori-Sato, C. et al. A conserved Mediator–CDK8 kinase module association regulates Mediator–RNA polymerase II interaction. Nat Struct Mol Biol 20, 611–619 (2013). https://doi.org/10.1038/nsmb.2549

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