Dissecting the stochastic transcription initiation process in liveEscherichia coli

Abstract

We investigate the hypothesis that, in Escherichia coli, while the concentration of RNA polymerases differs in different growth conditions, the fraction of RNA polymerases free for transcription remains approximately constant within a certain range of these conditions. After establishing this, we apply a standard model-fitting procedure to fully characterize the in vivo kinetics of the rate-limiting steps in transcription initiation of the Plac/ara-1 promoter from distributions of intervals between transcription events in cells with different RNA polymerase concentrations. We find that, under full induction, the closed complex lasts ∼788 s while subsequent steps last ∼193 s, on average. We then establish that the closed complex formation usually occurs multiple times prior to each successful initiation event. Furthermore, the promoter intermittently switches to an inactive state that, on average, lasts ∼87 s. This is shown to arise from the intermittent repression of the promoter by LacI. The methods employed here should be of use to resolve the rate-limiting steps governing the in vivo dynamics of initiation of prokaryotic promoters, similar to established steady-state assays to resolve the in vitro dynamics.

Temperature-Dependent Model of Multi-step Transcription Initiation in Escherichia coli Based on Live Single-Cell Measurements

“…Using statistical models, we identify the most likely changes with temperature in the rate limiting steps of transcription initiation. This expands on our previous work [22–24] in that the analysis at different temperatures allows identifying more complex underlying details of the kinetics of transcription. Also, we test if similar changes are observed in the P tetA promoter.…”

“…We compared our results on the relative durations of the pre- and post-commit steps with an independent method, namely, by extrapolating the corresponding values from a Lineweaver-Burk plot [24, 32]. For this, we used qPCR to measure the target gene activity and western blot to estimate the RNA polymerase concentrations in different media conditions (see S1 Appendix for details).…”

“…The last step in this model represents the complex escaping the start site, clearing the promoter region. Note that the reversibility of the closed complex formation [20, 24] does not reduce the applicability of the model [24, 25]. Also, regardless of this reversibility, the model can still be separated into an R -dependent (pre-commit) and R -independent (post-commit) parts.…”

“…To quantify intracellular RNA polymerase concentrations in each condition, we measured the amount of RpoC subunits by western blot (WB), as these are the limiting factor in the assembly of the RNA polymerase holoenzyme [39, 40]. Measuring gene activity as a function of the RNA polymerase abundance allows extrapolating the relative duration of the pre- and post-commit steps [24, 32]. …”

Transcription closed and open complex formation coordinate expression of genes with a shared promoter region

“…One aspect not yet considered is the existence of multiple rate-limiting steps during transcription initiation [14,15]. As only some of these steps are physically involved in the gene-pair interactions, we expect the nature of the rate-limiting steps of each promoter to affect the dynamics of closely-spaced configurations.…”