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Probing the architecture of the Mediator complex (939.3)
Author(s) -
Sierecki Emma,
Polinkovsky Mark,
Giles Nichole,
Moustaqil Mehdi,
Alexandrov Kirill,
Gambin Yann
Publication year - 2014
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.28.1_supplement.939.3
Subject(s) - mediator , multiprotein complex , protein subunit , transcription (linguistics) , rna polymerase ii , signal transducing adaptor protein , microbiology and biotechnology , biology , protein–protein interaction , repressor , transcription factor , transcription preinitiation complex , general transcription factor , computational biology , promoter , genetics , gene , gene expression , signal transduction , linguistics , philosophy
Gene transcription is an exquisitely regulated phenomenon in which multiple layers of protein‐protein interactions participate in the integration of the signal. The mechanism is mediated through interaction of transcription factors with a large array of regulatory proteins. “Activators” and “repressors” binding DNA upstream of the transcription factors provide positive or negative signals. Proteins called “co‐activators” or “adaptors” that cannot bind DNA by themselves, are responsible for tethering the transcription factors to the RNA polymerase and coordinate the influence of activators and repressors. These adaptors often form large protein assemblies. The largest complex, called “Mediator” is comprised of up to 25 subunits that are grouped into four modules called head, middle, CDK and tail, with a total mass of approximately 1 MDa. To study this adaptor protein complex, a rapid access to numerous recombinant subunits is required. Co‐expression of the subunits is also key to succeed as the level of disorder in subunits of Mediator is far greater than in other multiprotein complexes of similar size. Here, for the first time, we present a model for the architecture of the entire Mediator complex, obtained using a novel combination of protein analysis techniques. All possible subunit pairs were co‐expressed in a Leishmania tarentolae‐based cell‐free expression system and screened for their ability to interact by an AlphaScreen (Amplified Luminescent Proximity Homogeneous Assay Screen). Our results correlate with the published structure of the Mediator head and reveal intricate interactions between the subunits. Grant Funding Source : Supported by the ARC and NHMRC