Preparation and topology of the Mediator middle module

Koschubs, T.; Lorenzen, K.; Baumli, S.; Sandström, S.; Heck, A. J. R.; Cramer, P.

doi:10.1093/nar/gkq029

Item

ITEM ACTIONSEXPORT

Add to Basket

Local TagsRelease HistoryDetailsSummary

Released

Journal Article

Preparation and topology of the Mediator middle module

MPS-Authors

/persons/resource/persons127020

Cramer, P.
Department of Molecular Biology, MPI for Biophysical Chemistry, Max Planck Society;

External Resource

http://nar.oxfordjournals.org/content/38/10/3186.full.pdf
(Publisher version)

Fulltext (restricted access)

There are currently no full texts shared for your IP range.

Fulltext (public)

1938143.pdf
(Publisher version), 11MB

Supplementary Material (public)

1938143-Suppl-1.pdf
(Supplementary material), 2MB

1938143-Suppl-2.xls
(Supplementary material), 44KB

Citation

Koschubs, T., Lorenzen, K., Baumli, S., Sandström, S., Heck, A. J. R., & Cramer, P. (2010). Preparation and topology of the Mediator middle module. Nucleic Acids Research, 38(10), 3186-3195. doi:10.1093/nar/gkq029.

Cite as: https://hdl.handle.net/11858/00-001M-0000-0015-8326-C

Abstract

Mediator is the central coactivor complex required for regulated transcription by RNA polymerase (Pol) II. Mediator consists of 25 subunits arranged in the head, middle, tail and kinase modules. Structural and functional studies of Mediator are limited by the availability of protocols for the preparation of recombinant modules. Here, we describe protocols for obtaining pure endogenous and recombinant complete Mediator middle module from Saccharomyces cerevisiae that consists of seven subunits: Med1, 4, 7, 9, 10, 21 and 31. Native mass spectrometry reveals that all subunits are present in equimolar stoichiometry. Ion-mobility mass spectrometry, limited proteolysis, light scattering and small-angle X-ray scattering all indicate a high degree of intrinsic flexibility and an elongated shape of the middle module. Protein–protein interaction assays combined with previously published data suggest that the Med7 and Med4 subunits serve as a binding platform to form the three heterodimeric subcomplexes, Med7N/21, Med7C/ 31 and Med4/9. The subunits, Med1 and Med10, which bridge to the Mediator tail module, bind to both Med7 and Med4.