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Nat Struct Mol Biol. 2014 Dec;21(12):1028-34. doi: 10.1038/nsmb.2914. Epub 2014 Nov 10.

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

Author information

1
Laboratory of Biochemistry and Molecular Biology, Rockefeller University, New York, New York, USA.
2
Laboratory of Mass Spectrometry and Gaseous Ion Chemistry, Rockefeller University, New York, New York, USA.

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.

PMID:
25383669
PMCID:
PMC4259101
DOI:
10.1038/nsmb.2914
[Indexed for MEDLINE]
Free PMC Article

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