Simple biochemical features underlie transcriptional activation domain diversity and dynamic, fuzzy binding to Mediator (Activation)

A pooled screen for protein fragments that induce transcriptional activation of a reporter GFP gene. Overall design: Protein fragments of length 53 amino acids were synthesized as an oligo pool and cloned into a plasmid library. Outgrowth of transformed yeast for five days ensured that each cell contained a unique aTF expression plasmid due to mechanisms maintaining it at single-copy levels. After induction with estrogen, cells with a defined level of aTF expression were selected by mCherry signal and sorted into eight bins based on GFP expression. By next-generation sequencing, the distribution of each protein tile across the bins was determined and the mean value was used to calculate activation—namely, the fold increase in GFP relative to background