Figure 6. Synthetic ZF-Based Transcription Framework Can Be Used to Engineer Diverse Two-Input Behaviors.
(A) The transcriptional operation of a single sTF43-8 (carrying a PDZ domain) at the proximal position of a two-input promoter.
(B) Cooperative two-input synergy engineered with PDZ-carrying sTF43-8-4x as the proximal activator and cognate ligand-carrying sTF42-10-4x as the distal partner.
(C) Cooperative two-input synergy further enhanced by the addition of an AD onto the distal partner to create a two-activator system.
(D) A “null” two-input system engineered by abolishing the dimerization interaction with a PDZ nonbinding ligand on the distal partner, thus rendering it noncontributory.
(E and F) Inhibitory two-input behavior engineered by reversing the activator location (from proximal to distal) and using either PDZ binding (E) or nonbinding ligands (F).
(G and H) Inhibition by the proximal monomer can be further increased by increasing the proximal ZF affinity to DNA (43-8-4x to 43-8-3x) and decreasing the distal ZF affinity to DNA (42-10-3x to 42-10-4x) in both PDZ binding (G) and nonbinding cases (H).
(I) By reversing the orientation of the operators, sTF43-8-4x is converted from an inhibitor to a cooperative factor to, once again, obtain cooperative transcriptional synergy in the two-input behavior. All sTFs were expressed from either ATc- or IPTG-inducible pGAL1 (500 ng/ml ATc and/or 20 mM IPTG). Horizontal axes correspond to “mean fluorescence intensity per cell (AU)” and begin at basal (promoter-only) fluorescence level. Error bars represent SD of three experiments.