Measuring cis-regulatory energetics in living cells using allelic manifolds

Gene expression in all organisms is controlled by cooperative interactions between DNA-bound transcription factors (TFs), but quantitatively measuring TF-DNA and TF-TF interactions remains difficult. Here we introduce a strategy for precisely measuring the Gibbs free energy of such interactions in living cells. This strategy centers on the measurement and modeling of ‘allelic manifolds’, a multidimensional generalization of the classical genetics concept of allelic series. Allelic manifolds are measured using reporter assays performed on strategically designed cis-regulatory sequences. Quantitative biophysical models are then fit to the resulting data. We used this strategy to study regulation by two Escherichia coli TFs, CRP and [Formula: see text] RNA polymerase. Doing so, we consistently obtained energetic measurements precise to [Formula: see text] kcal/mol. We also obtained multiple results that deviate from the prior literature. Our strategy is compatible with massively parallel reporter assays in both prokaryotes and eukaryotes, and should therefore be highly scalable and broadly applicable. Editorial note: This article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that minor issues remain unresolved (see decision letter).