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.
Submitter: Max Staller
Submission Date: July 10, 2018, 8:25 a.m.
|Number of data points||21899|
|Proteins||General control protein GCN4 -Central acidic activation domain (101-144)|
|Assays/Quantities/Protocols||Experimental Assay: Activation domain variant abundance ; Experimental Assay: Activation domain induction ; Experimental Assay: Activation domain activity|
|Libraries||Gcn4 activation domain mutants - protein abundance ; Gcn4 activation domain mutants - protein abundance ; Gcn4 activation domain mutants -Induction ; Gcn4 activation domain mutants - Activity Replicate 1 ; Gcn4 activation domain mutants - Activity Replicate 2|