Measuring cis-regulatory energetics in living cells using allelic manifolds
Measuring cis-regulatory energetics in living cells using allelic manifolds
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DOI：
10.7554/elife.40618
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发表时间：
2018-12-20
期刊：
影响因子：
7.7
通讯作者：
Kinney, Justin B.
中科院分区：
文献类型：
--
作者：
Forcier, Talitha L.;Ayaz, Andalus;Kinney, Justin B.
在所有生物中，基因的表达都是由DNA结合的转录因子之间的协同作用控制的，但定量测量转录因子-DNA和转录因子-转录因子的相互作用仍然是困难的。在这里，我们介绍了一种精确测量活细胞中这种相互作用的吉布斯自由能的策略。这一策略的核心是“等位基因流形”的测量和建模，这是等位基因序列的经典遗传学概念的多维概括。等位基因流形是通过对策略性设计的顺式调控序列进行的报告分析来测量的。然后将定量的生物物理模型与所得到的数据进行拟合。我们利用这一策略研究了两种大肠杆菌转录因子--CRP和Sigma(70)RNA聚合酶的调控。这样做，我们始终获得精确到0.1千卡/摩尔的能量测量。我们还得到了与先前文献相背离的多个结果。我们的策略与原核生物和真核生物的大规模平行报告分析兼容，因此应该具有高度的可扩展性和广泛的适用性。编辑注：本文经过编辑过程，作者决定如何回应同行审查中提出的问题。审查编辑的评估是，次要问题仍未得到解决(见决定函)。
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 sigma(70) RNA polymerase. Doing so, we consistently obtained energetic measurements precise to similar to 0.1 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).