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Uncovering transcription factor binding pockets in the tail subdomain of the human mediator complex (943.2)
Author(s) -
Polinkovsky Mark,
Sierecki Emma,
Giles Nichole,
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.943.2
Subject(s) - mediator , transcription factor , protein subunit , biology , transcription (linguistics) , protein–protein interaction , taf2 , microbiology and biotechnology , general transcription factor , plasma protein binding , genetics , binding site , transcriptional regulation , computational biology , gene , gene expression , promoter , enhancer , linguistics , philosophy
The Mediator Complex plays a major role in regulating gene transcription in eukaryotes. One of its fundamental roles is to integrate inputs from a variety of transcription factors to control the gene’s transcriptional state. The majority of these transcription factors are thought to bind to the Mediator’s Tail subdomain. In humans, the Tail is composed of eight subunit proteins, whose structure and arrangement remain unknown, largely due to difficulties in expressing and purifying them. Moreover, only a few interactions between transcription factors and some of the Tail subunits have been found. To overcome difficulties in protein expression and interaction analysis, we used the Leishmania cell‐free protein expression system and pair‐wise measurements of protein interactions via the AlphaScreen assay. These methods allowed us to build a model of the interactions between protein subunits in the Mediator Tail. As validation, we pulled down all the component proteins in the Tail subdomain from a single cell‐free co‐expression. The same techniques were used to test the binding of various transcription factors to the Tail subunits. Our results show that different families of transcription factors bind to distinct groups of subunits in the Tail. Combined with our model of the Tail, this data reveals binding pockets on the Tail’s surface. Grant Funding Source : Supported by Australian Research Council