Effects of σ factor competition are promoter initiation kinetics dependent.

In Escherichia coli, the expression of a σ factor is expected to indirectly down-regulate the expression of genes recognized by another σ factor, due to σ factor competition for a limited pool of RNA polymerase core enzymes. Evidence suggests that the sensitivity of genes to indirect down-regulation differs widely. We studied the variability in this sensitivity in promoters primarily recognized by RNAP holoenzymes carrying σ(70). From qPCR and live single-cell, single-RNA measurements of the transcription kinetics of several σ(70)-dependent promoters in various conditions and from the analysis of σ factors population-dependent models of transcription initiation, we find that, the smaller is the time-scale of the closed complex formation relative to the open complex formation, the weaker is a promoter's responsiveness to changes in σ(38) numbers. We conclude that, in E. coli, a promoter's responsiveness to indirect regulation by σ factor competition is determined by the sequence-dependent kinetics of the rate limiting steps of transcription initiation.

Dissecting the stochastic transcription initiation process in live Escherichia coli.

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.