A High-Throughput Mutational Scan of an Intrinsically Disordered Acidic Transcriptional Activation Domain

Transcriptional activation domains are essential for gene regulation, but their intrinsic disorder and low primary sequence conservation have made it difficult to identify the amino acid composition features that underlie their activity. Here, we describe a rational mutagenesis scheme that deconvolves the function of four activation domain sequence features—acidity, hydrophobicity, intrinsic disorder, and short linear motifs—by quantifying the activity of thousands of variants in vivo and simulating their conformational ensembles using an all-atom Monte Carlo approach. Our results with a canonical activation domain from the Saccharomyces cerevisiae transcription factor Gcn4 reconcile existing observations into a unified model of its function: the intrinsic disorder and acidic residues keep two hydrophobic motifs from driving collapse. Instead, the most-active variants keep their aromatic residues exposed to the solvent. Our results illustrate how the function of intrinsically disordered proteins can be revealed by high-throughput rational mutagenesis. Since their discovery over 30 years ago, it has been unclear why many transcriptional activation domains of transcription factors are acidic. Staller et al. describe a method to measure the activities of thousands of rationally designed activation domains’ mutants. With this method, they uncover a role for the acidic residues of a classic model activation domain.