Journal: eLife
Article Title: Simple biochemical features underlie transcriptional activation domain diversity and dynamic, fuzzy binding to Mediator
doi: 10.7554/eLife.68068
Figure Lengend Snippet: ( A ) We took 47 pairs of adjacent cADs and measured their activation enhancement factors—the activation of both cADs in tandem divided by the product of activation by each cAD individually. For 40 pairs, activation was enhanced when cADs were in tandem, with a median enhancement factor of 4.0-fold. ( B ) Kinetics of Mediator complex recruitment to DNA by Gcn4. ( Top ) We coupled DNA containing a Gcn4 motif to a NeutrAvidin-coated surface, added Gcn4, and measured real-time binding of Mediator by surface plasmon resonance. The Mediator-binding step also included 7.5 nM Gcn4 to maintain DNA-bound Gcn4. ( Bottom ) Real-time binding of 2.5 nM Mediator to Gcn4-DNA complexes (0 to 480 seconds) and subsequent dissociation (480 seconds onwards). DNA templates contained 0, 1, 2, 4, or 6 copies of the Gcn4 motif. AU, arbitrary units. ( C ) The interaction half-life of Mediator with Gcn4-DNA complexes was proportional to the number of Gcn4 motifs and independent of the concentration of Mediator used in the binding step. See also . ( D ) Gcn4 competition assay. We purified a fusion of the Gcn4 AD to nuclease deficient EcoRI(E111Q), which resides on DNA for several hours, bound it to DNA containing a single EcoRI site, and measured Mediator recruitment in the presence or absence of excess Gcn4 competitor. Mediator was at 5 nM in all six conditions. AU, arbitrary units. See also . ( E ) Model of Mediator-TF interaction that explains high-affinity but dynamic binding. Multiple weak, short-lived interactions between individual Mediator ABDs (blue) and TF ADs (maroon) together drive longer-lived high-affinity binding. However, rapid association-dissociation equilibrium of an AD allows a second Mediator molecule to interact with Mediator-bound TF, facilitating the dynamic exchange of one Mediator molecule by another. The same mechanism would allow exchange of one TF by another. ( F ) An activation screen of nuclear proteins identified ADs in all major coactivator and chromatin modifying complexes. Protein complex, protein name, and start position of the 53-aa AD is labeled, and experimentally measured activation (green) and fraction of residues predicted to be disordered (purple; in D2P2) is shown. ADs that are predominantly disordered or unresolved in PDB structures are displayed in purple text. Taf10 is also a subunit of SAGA and Arp4 is also part of the INO80 chromatin remodeling complex. See also and .
Article Snippet: SPR experiments were performed on the ProteOn XPR36 Surface Plasmon Resonance System using NeutrAvidin-coated NLC Sensor Chips (Bio Rad).
Techniques: Activation Assay, Binding Assay, SPR Assay, Concentration Assay, Competitive Binding Assay, Purification, Labeling
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