SuperTrxn

This work investigates supercoil-mediated transcription regulation in bacteria by an integrated approach encompassing in vitro, in vivo, and in silico studies. Recent work suggests that the supercoiling state (under- or over-winding) of DNA can influence transcription kinetics and modulate the correlation between multiple RNA polymerase (RNAP) molecules within and between neighboring transcription units (TUs). As transcription also generates supercoils, the complex feedback between supercoils and transcription introduces a novel layer of DNA-based regulation beyond classic protein-based regulation in many organisms. In chromosomal topological domains, supercoils may thus couple the expression dynamics of neighboring TUs and influence gene expression and cell physiology. While remarkable progress has been made to establish the effect of supercoils on transcription, a quantitative description of their role in regulating transcription kinetics and correlative transcription dynamics remains elusive. In this work, the US and French partners will combine their expertise in experimental investigations of bacterial transcription and theoretical modeling of DNA supercoiling and transcription to address three objectives: (1) characterize how supercoiling impacts transcription kinetics and TU correlation using in vitro single-molecule imaging; (2) characterize how chromosomal topological domains affect gene correlation using in vivo single-molecule imaging; (3) model and simulate supercoiling-mediated transcription regulation at all molecular and time scales of transcription. This work will provide significant knowledge about a new transcription regulation mechanism independent of transcription factor binding and unbinding. (1) It will produce sensitive transcription kinetics measurements, the temporal dynamics of the supercoil-mediated interplay between neighboring RNAP molecules and TUs, and the impact of the chromosomal topological domain on these kinetics and dynamics. These quantitative and systematic measurements are only made possible by innovative single-molecule imaging in vitro and in vivo. (2) This work will develop a comprehensive theoretical framework describing quantitative supercoil-mediated transcription regulation, from DNA base-pair energetics to kilobase propagation of supercoils, and predictive of the coordination/antagonism of neighboring RNAP molecules and TUs depending on topological domain formation and topoisomerase activities. (3) The close integration between experiments and modeling enables iterative model calibration and refinements, which provide further guidance for experimental design, testing, and interpretation. If successfully carried out, this study will advance substantially the field of bacterial transcription regulation and shed light on how chromosomal topology quantitatively regulates gene expression.