Reconstitution of active human core Mediator complex reveals a critical role of the MED14 subunit.
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.
Laboratory of Biochemistry and Molecular Biology, Rockefeller University, New York, New York, USA.
Murat A
MA
Cevher
Laboratory of Mass Spectrometry and Gaseous Ion Chemistry, Rockefeller University, New York, New York, USA.
Yi
Y
Shi
Laboratory of Biochemistry and Molecular Biology, Rockefeller University, New York, New York, USA.
Dan
D
Li
Laboratory of Mass Spectrometry and Gaseous Ion Chemistry, Rockefeller University, New York, New York, USA.
Brian T
BT
Chait
Laboratory of Biochemistry and Molecular Biology, Rockefeller University, New York, New York, USA.
Sohail
S
Malik
Laboratory of Biochemistry and Molecular Biology, Rockefeller University, New York, New York, USA.
Robert G
RG
Roeder
D003001:Cloning, Molecular; D006367:HeLa Cells; D006801:Humans; D056892:Mediator Complex / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D060066:Protein Interaction Maps; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012319:RNA Polymerase II / Q000378:metabolism; D015533:Transcriptional Activation
C492160:MED14 protein, human; D056892:Mediator Complex; D021122:Protein Subunits; D012319:RNA Polymerase II