Nucleosomal Proofreading of Activator-Promoter Recognition

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This research program investigates the role of random molecular behavior in regulating gene expression by "transcriptional activators", molecules that control the activity of genes by binding to specific regulatory sequences in DNA. The project introduces students from diverse backgrounds to a fundamental theoretical problem at the interface between molecular genetics and statistical physics as well as advanced experimental tools to address this problem. Pedagogically, the program aims to integrate mathematical, physical, and quantitative reasoning into the biology curriculum for undergraduate and graduate students and provide research opportunities to high school students to increase participation in STEM fields.The specificity of transcriptional regulation – which genes are transcribed in response to a particular signal – is thought to rest entirely with the specificity of activator-gene recognition. Yet, for many eukaryotic activators the energetic differences between correct and incorrect sequence binding are remarkably small – too small to explain observed regulatory specificities. The specificity problem may be solved by "kinetic proofreading", i.e., insertion of a free energy-transducing delay step between activator-promoter binding and transcription. The mechanism enhances specificity not by increasing the energetic difference between correct and incorrect associations but by exploiting the same difference twice, before the delay step and afterward, when free energy dissipation favors complex dissociation over formation and return of the suspended delay mechanism to its repressive state. A prime candidate among possible delay mechanisms for activator proofreading is ATP-dependent chromatin remodeling, which relieves genes from nucleosomal repression in an activator-dependent manner. Logical implications of this proposition, including the prediction that perturbation of chromatin remodeling affects the ability of activators to distinguish between correct and incorrect genes, will be tested by employing a combination of live-cell multifocus fluorescence microscopy, single molecule-fluorescence in situ hybridization, nanopore sequencing, and reverse genetics in yeast. The outcomes will provide new insights on how near deterministic behavior of biological systems emerges from the random behavior of their molecular components.This project is jointly funded by the Genetic Mechanisms program of the Molecular and Cellular Biosciences Division in the Biological Sciences Directorate and the Mathematical Biology program of the Mathematical Sciences Division in the Mathematical and Physical Sciences Directorate.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.