A Cooperative Mechanism of Eukaryotic Transcription Factor Target Search

Rapid gene activation requires transcription factors (TFs) to locate their target motifs within vast genomes. In bacteria, TF target search is accelerated by combining 3D diffusion with 1D sliding, called facilitated diffusion, yet whether eukaryotic TFs rely on similar strategies has remained unresolved due to the lack of direct measurements in living cells. Here, we directly visualize eukaryotic TF target search in living cells by labeling a single TF per nucleus and visualizing its binding to its endogenous locus. Using the budding yeast TF Gal4, we find that efficient target localization occurs near the diffusion limit and does not require facilitated diffusion. Instead, rapid association requires cooperative self-interactions mediated by an intrinsically-disordered central region (IDR), independent of the activation domain. Replacing the Gal4 IDR with human self-interacting IDRs (EWS or FUS) restores efficient search, demonstrating that self-interactions are a general and portable feature for search. A second structured dimerization domain further cooperatives with the IDR to stabilize binding at neighboring motifs, revealing two mechanistically separable forms of cooperativity that together govern TF function. These findings highlight that facilitated diffusion is not strictly required and establish cooperative IDR-driven self-interactions as a key mechanism to enable rapid target recognition in eukaryotic cells.