Preparation and topology of the Mediator middle module.

Nucleic Acids Research
Tobias KoschubsPatrick Cramer

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.

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Citations

Jun 17, 2011·Journal of the American Chemical Society·Linjie HanBrandon T Ruotolo
Dec 20, 2011·Molecular & Cellular Proteomics : MCP·Florian StengelCarol V Robinson
Jun 28, 2012·PLoS Computational Biology·Theresa NiederbergerAchim Tresch
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Feb 9, 2019·Biochemical Society Transactions·Alexis VergerVincent Villeret

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Methods Mentioned

BETA
X-ray
nuclear magnetic resonance
two-hybrid
tandem affinity purification
PCR
gel filtration
gel
pull-down
two
immunoprecipitation

Software Mentioned

CDM
HHpred
Tasser
Drift Scope
PSIpred
IM
ATSAS

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