Nucleosomes and IDRs suppress promiscuous GCN4 binding on minichromosomes

Eukaryotic sequence-specific transcription factors (TFs) must find their cognate DNA targets hidden in genomic chromatin amid an excess of nonspecific sequences and degenerate motifs. Although static TF interactions with nucleosomal targets have been elucidated, how TFs efficiently search for cognate sites within native gene-sized chromatin domains has been unclear. Here we used purified Saccharomyces cerevisiae HIS3 minichromosomes and single-molecule imaging to compare association and dissociation kinetics of transcription activator GCN4 on chromatin and naked genomic DNA. GCN4 displays widespread and stable off-target binding on bare DNA because of entrapment by degenerate sites and interactions with nonspecific DNA of increasing length, indicative of one-dimensional (1D) diffusion. Nucleosome organization on the minichromosome reduces promiscuous GCN4 residence times by obstructing TF association and restricting 1D target search within nucleosome-free regions. Furthermore, the intrinsically disordered GCN4 activation domain independently enhances targeting efficiency and specificity by accelerating association-dissociation kinetics in vitro and in living cells. Altogether, both nucleosome organization and activation domains independently suppress promiscuous GCN4 binding, which, if unchecked, may cause aberrant cryptic transcription known to occur upon chromatin disruptions.