Investigating RNAP Search Dynamics in Live E. Coli Cells using Single Molecule and Statistical Methods

Abstract

Transcription is an essential component of gene regulation and one of the most highly regulated processes in the cell. Because bacteria lack a nuclear envelope, genes transcribed by RNA polymerase (RNAP), the key enzyme of transcription, are essentially immediately translated into protein, leaving the kinetics of transcription significantly influencing the resulting protein expression timing and levels. However, while the kinetics of transcription is greatly studied in vitro, the in vivo dynamics of RNAP, including the typical search time and transcription time, are difficult to obtain. Our lab takes advantage of the sensitivity and fast time scale of single-molecule tracking to determine the kinetics of RNAP binding and transcription in live E. coli cells. We used a strain of E. coli in which the β’ subunit (rpoC) of RNAP is tagged with a photoactivatable fluorescent protein, PAmCherry, replacing the endogenous chromosomal copy. We discovered there are three states of RNAP with distinct diffusion coefficients. Using genetic manipulations and drug treatments, we were able to assign each diffusion state to a particular function of RNAP, i.e. RNAP that is bound to the DNA, RNAP that is undergoing rapid association and dissociation from the nucleoid, and freely diffusing RNAP. Further, using a Markov chain Monte Carlo algorithm, we were able to form a hidden Markov model of our data to parse out the dynamics of RNAP switching states, which enabled us to parse out a complete dynamical model of RNAP in living cells. We obtainined a wide variety of important parameters, including the non-specific dwell time, the typical promoter search time, and the typical transcription time for an average gene. These parameters will be useful for future gene regulations studies of all kinds, as well as to help understand general protein-nucleic acid interactions and dynamics within the crowded cytoplasm of a live cell.