Diffusion is at the heart of every biochemical process. Millions of proteins must navigate the heterogeneous, crowded cellular milieu to perform their various tasks. This molecular crowding has a significant effect on the diffusive behavior and kinetic rates of proteins and biochemical reactions. A powerful technique to understand biochemical processes within the context of this heterogeneous environment is single particle tracking (SPT). In Chapter 2, I use SPT to elucidate the dynamics of the RNAP search process and transcription cycle in live E. coli cells. Using FRAP, I find that transcription follows a simple initiation-elongation-termination cycle with kinetic rates that closely match those in the literature. Using SPT, I probed the search process of RNAP and found three diffusive states corresponding to DNA-bound, diffusion within the dense nucleoid, and diffusion within the cytoplasm. RNAP exhibited confinement in each state and displayed a preference for a DNA-bound state, suggesting a grid search strategy. Additionally, RNAP displayed kinetics that were not consistent with steady state kinetics. In Chapter 3, I use SPT to probe the molecular mechanism of HU-mediated chromosome organization. Using genetic mutations that abolish the various binding modes of HU, I find that HUαα and HUαβ displayed differential dynamics. Additionally, HUαα seems primarily responsible for non-specific binding while HUαβ seems primarily responsible for repressor loop formation. The kinetics of HU were highly transient, indicative of their non-specific binding across the nucleoid, and suggested a mechanism by which cumulative forces of thousands of HU are able to achieve chromosomal organization, a marked departure from the long-lived binding of other DNA organization proteins such as histones
Electronic reproduction. Ann Arbor, Mich. : ProQuest, 2020
