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Precision measurement of cis-regulatory energetics in living cells

Forcier, Talitha and Ayaz, Andalus and Gill, Manraj S. and Jones, Daniel and Phillips, Rob and Kinney, Justin B. (2018) Precision measurement of cis-regulatory energetics in living cells. . (Unpublished)

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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.

Item Type:Report or Paper (Discussion Paper)
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URLURL TypeDescription Paper
Jones, Daniel0000-0002-7902-996X
Phillips, Rob0000-0003-3082-2809
Kinney, Justin B.0000-0003-1897-3778
Additional Information:The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-ND 4.0 International license. We thank Bryce Nickels and Stirling Churchman for helpful feedback. This work was supported by a CSHL/Northwell Health Alliance grant to JBK and by NIH Cancer Center Support Grant 5P30CA045508. Author contributions: JBK conceived of this study. TF and JBK designed this study. JBK, TF, AA, MSG, and DJ carried out the experiments. TF and JBK carried out the computational analysis. JBK wrote the manuscript with input from MSG, RP, DJ, TF, and AA. JBK funded this study.
Funding AgencyGrant Number
Cold Spring Harbor Laboratory/Northwell Health AllianceUNSPECIFIED
Record Number:CaltechAUTHORS:20180927-114222775
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Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:89992
Deposited By: George Porter
Deposited On:27 Sep 2018 23:21
Last Modified:16 Nov 2021 00:40

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