Investigating the biophysical mechanisms regulating transcription in starved Escherichia coli via single-molecule tracking.

Abstract

Bacteria have a robust stress response in starvation conditions, entering a quiescent stationary phase and remaining viable despite depletion of resources. An important component of this stress response is the upregulation of DNA-binding protein from starved cells (Dps) upon entry into the stationary phase. Dps promotes nucleoid condensation, protecting genetic information from harm. Intriguingly, this condensation does not affect the ability of RNA polymerase (RNAP) to access the nucleoid and transcribe RNA. The mechanism by which RNAP accesses the nucleoid under starvation conditions remains unclear. To investigate the dynamics of stationary-phase RNAP, we are applying live-cell single-molecule tracking of RNAP tagged with a photo-activatable fluorescent protein. In living E. coli cells, we find that compared to the dynamics of RNAP in the exponential growth phase, RNAP in the stationary phase spends less time bound to DNA, reflective of an expected decline in protein synthesis. Instead, stationary-phase RNAP spends the majority of its time in an intermediate dynamic state that we hypothesize results from frequent interactions with DNA as RNAP searches for its target sequence. Unexpectedly, the diffusion coefficient of this intermediate state increases in the stationary phase, even though the nucleoid is more compact and thus has a smaller pore size that could restrict motion. To isolate how Dps upregulation leads to changes in the dynamics of RNAP throughout the cell cycle, we over-expressed Dps and found that the RNAP dynamics were nearly identical to those measured with native Dps expression. Ultimately, these studies suggest that the interactions between RNAP and DNA are primarily biochemically driven, and do not depend on nucleoid pore size. Additionally, our results may indicate that Dps can be rapidly displaced by RNAP such that it does not interfere with target searching.