Collaborative Research: NSF/MCB: Kinetic Control of the Transcription Cycle Revealed by Synthetic Enhancers

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Genes encode molecular tools in the form of RNA and protein. To use these tools to respond to signals and execute different behaviors, cells carefully control when, where and how much genes are turned on. This is called 'gene regulation'. A major goal of synthetic biology is to engineer cells to perform specific useful tasks. To accomplish this goal, researchers must learn how gene regulation works well enough to predict and build genes that will be turned on in exactly the right place, at the right time and to the right level. This project explores the idea that gene regulation is a multi-step cycle that is regulated at specific points, like driving a car. A car contains many small interacting parts that have to act in a specific order to produce force and move, but to drive it, one only need to operate the ignition, the gas, the steering wheel and the brakes. The investigators in the project are trying to identify which steps are regulated, and which molecules regulate them. The work will contribute to development of new tools that will advance biomanufacturing, enable the design of new drugs to treat disease, and will provide new methods for enhancing crop yield, and other applications that will benefit from the ability to precisely control biology and biotechnology.For decades, transcription research has been driven by a model that is conceptually simple: proteins bind to regulatory DNA at equilibrium to regulate recruitment of RNA polymerase (RNAP). This has led to a focus on cooperative and competitive physical interactions between transcription factors (TFs), cofactors and the basal transcriptional machinery, and underlies the search for the still hypothetical 'cis-regulatory code' of TF binding sites. This project challenges this dominant paradigm of transcriptional control by addressing how kinetic processes could combinatorially regulate transcription. Because promoters dictate rate limiting steps for transcription, we will measure the number of slow rates at multiple promoters using high resolution imaging, and test whether this rate spectrum is sensitive to cell type (Aim 1). TFs accelerate or retard different parts of this complex, dynamic cycle through their distinct biochemical activities. This project will use functional genomics and knock-down to characterize TF function at endogenous genes and at synthetic enhancers (Aim 2). The long-term goal is to predict and build synthetic gene regulatory systems that will both illuminate fundamental biology and perform useful functions, by performing quantitative experiments that are contextualized with theory based in biophysics.