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

• Published in: Cell systems Vol. 6; no. 4; pp. 444 - 455.e6
• Main Authors: Staller, Max V., Holehouse, Alex S., Swain-Lenz, Devjanee, Das, Rahul K., Pappu, Rohit V., Cohen, Barak A.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 25.04.2018
• Subjects: all-atom simulations; Basic-Leucine Zipper Transcription Factors - chemistry; Basic-Leucine Zipper Transcription Factors - physiology; Catalytic Domain; deep mutational scanning; Gene Expression Regulation; gene regulation; genomic method development; high-throughput mutagenesis; Hydrogen-Ion Concentration; intrinsically disordered protein; intrinsically disordered region; Models, Molecular; Monte Carlo Method; Mutagenesis, Site-Directed; Protein Domains; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae Proteins - chemistry; Saccharomyces cerevisiae Proteins - physiology; Sequence Analysis, Protein; transactivation domain; transcription factor activation domain; transcription factors; Transcription Factors - chemistry; Transcription Factors - physiology; all-atom simulations; intrinsically disordered protein; deep mutational scanning; transcription factors; gene regulation; high-throughput mutagenesis; intrinsically disordered region; transactivation domain; genomic method development; transcription factor activation domain
• ISSN: 2405-4712; 2405-4720
• DOI: 10.1016/j.cels.2018.01.015

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. [Display omitted] •A high-throughput method for measuring the strength of activation domain mutants•The central activation domain of Gcn4 becomes stronger during amino acid starvation•Highly active Gcn4 mutants keep aromatic residues exposed to solvent•Acidic residues create a permissive context for a hydrophobic short linear motif 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.