NSF-ANR MCB/PHY SuperTrxn In vitro, in vivo, and in silico investigations of supercoil-mediated transcription regulation in bacteria

This project investigates how supercoiled DNA impacts transcription, a core process of life that converts genetic information encoded in DNA to RNA. Classical models of transcription regulation depict that proteins such as transcription factors bind specific DNA sequences to turn a gene on or off. Recent studies suggest that the mechanics of DNA itself, such as supercoiling, the over- or under-twisting of a double-stranded DNA, can influence how RNA polymerase (RNAP) transcribes RNA. As such, supercoiling could act as a transcription regulator and play a vital role in modulating gene expression in cells. We will examine this hypothesis by performing imaging experiments both inside and outside cells and developing computational models of the process. The work will provide significant knowledge about a new transcription regulation mechanism independent of transcription factor activity. The interdisciplinary nature of the research will also provide excellent research training and teaching opportunities for undergraduate and graduate students. The project will also support participation of Baltimore city public school children in science camps. The research will (1) characterize how supercoiling impacts transcription kinetics and correlation in vitro using single-molecule imaging of purified protein and DNA constructs; (2) characterize how chromosomal topological domains impact gene expression in vivo using single-molecule fluorescence in situ hybridization (smFISH) and single-molecule gene expression reporters; and (3) model and simulate transcriptional regulation by supercoiling at all molecular and time scales of transcription by combining coarse-grained modeling of DNA base-pairing under torque and data obtained from the experiments. The research is anticipated to yield sensitive transcription kinetics measurements that reveal the temporal dynamics of supercoil-mediated interplay between neighboring RNAP molecules and genes and the impact of chromosomal topological domains on these kinetics and dynamics. The overarching goal is to develop a comprehensive quantitative theoretical framework describing supercoil-mediated transcription regulation, from DNA base-pair energetics to kilobase propagation of supercoils. This collaborative US/France project is jointly supported by the Genetic Mechanisms program in the Division of Molecular and Cellular Biosciences and the Physics of Living Systems program in the Division of Physics at the US National Science Foundation and the French Agence Nationale de la Recherche, where NSF funds the US investigator and ANR funds the partners in France.” 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.