title: Preparation and topology of the Mediator middle module.
abstract: Mediator is the central coactivator 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.
authors.affiliation: Department of Biochemistry, Gene Center Munich and Center for Integrated Protein Science Munich, Ludwig-Maximilians-Universität München, Feodor-Lynen-St. 25, 81377 Munich, Germany.
authors.forename: Tobias
authors.initials: T
authors.lastname: Koschubs
authors.forename: Kristina
authors.initials: K
authors.lastname: Lorenzen
authors.forename: Sonja
authors.initials: S
authors.lastname: Baumli
authors.forename: Saana
authors.initials: S
authors.lastname: Sandström
authors.forename: Albert J R
authors.initials: AJ
authors.lastname: Heck
authors.forename: Patrick
authors.initials: P
authors.lastname: Cramer
mesh_terms: D056892:Mediator Complex / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D056892:Mediator Complex; D021122:Protein Subunits; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins