A sequence-specific transcription activator motif and powerful synthetic variants that bind Mediator using a fuzzy protein interface

Significance How transcription activators recognize their coactivator targets is a longstanding question and is important for understanding activator specificity and synergy. Most activators are not obviously related in sequence, but they recognize a common set of coactivators, raising the question of whether these interactions are sequence-specific. We show that the yeast transcription factor Gcn4 central activation domain works via a short sequence-specific motif that can be optimized to generate powerful synthetic activators. Like many natural activators, the synthetic derivatives have redundant sequence and bind the Mediator subunit Gal11 with high affinity using a “fuzzy” protein interface. Our results suggest a mechanism to explain how a subset of natural activators use redundant sequence motifs and great flexibility in the binding interface to target unrelated coactivators. Although many transcription activators contact the same set of coactivator complexes, the mechanism and specificity of these interactions have been unclear. For example, do intrinsically disordered transcription activation domains (ADs) use sequence-specific motifs, or do ADs of seemingly different sequence have common properties that encode activation function? We find that the central activation domain (cAD) of the yeast activator Gcn4 functions through a short, conserved sequence-specific motif. Optimizing the residues surrounding this short motif by inserting additional hydrophobic residues creates very powerful ADs that bind the Mediator subunit Gal11/Med15 with high affinity via a “fuzzy” protein interface. In contrast to Gcn4, the activity of these synthetic ADs is not strongly dependent on any one residue of the AD, and this redundancy is similar to that of some natural ADs in which few if any sequence-specific residues have been identified. The additional hydrophobic residues in the synthetic ADs likely allow multiple faces of the AD helix to interact with the Gal11 activator-binding domain, effectively forming a fuzzier interface than that of the wild-type cAD.

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