PT  - JOURNAL ARTICLE
AU  - Talitha Forcier
AU  - Andalus Ayaz
AU  - Manraj S. Gill
AU  - Daniel Jones
AU  - Rob Phillips
AU  - Justin B. Kinney
TI  - Precision measurement of cisregulatory energetics in living cells
AID  - 10.1101/380972
DP  - 2018 Jan 01
TA  - bioRxiv
PG  - 380972
4099  - http://biorxiv.org/content/early/2018/07/31/380972.short
4100  - http://biorxiv.org/content/early/2018/07/31/380972.full
AB  - Gene expression in all organisms is controlled by cooperative interactions between DNA-bound transcription factors (TFs). However, measuring TF-TF interactions that occur at individual cis-regulatory sequences remains difficult. Here we introduce a strategy for precisely measuring the Gibbs free energy of such interactions in living cells. Our strategy uses reporter assays performed on strategically designed cis-regulatory sequences, together with a biophysical modeling approach we call “expression manifolds”. We applied this strategy in Escherichia coli to interactions between two paradigmatic TFs: CRP and RNA polymerase (RNAP). Doing so, we consistently obtain measurements precise to ~ 0.1 kcal/mol. Unexpectedly, CRP-RNAP interactions are seen to deviate in multiple ways from the prior literature. Moreover, the well-known RNAP binding motif is found to be a surprisingly unreliable predictor of RNAP-DNA binding energy. Our strategy is compatible with massively parallel reporter assays in both prokaryotes and eukaryotes, and should thus be highly scalable and broadly applicable.