Temperature-dependence of the single-cell variability in the kinetics of transcription activation inEscherichia coli

Abstract

From in vivo single-cell, single-RNA measurements of the activation times and subsequent steady-state active transcription kinetics of a single-copy Lac-ara-1 promoter in Escherichia coli, we characterize the intake kinetics of the inducer (IPTG) from the media, following temperature shifts. For this, for temperature shifts of various degrees, we obtain the distributions of transcription activation times as well as the distributions of intervals between consecutive RNA productions following activation in individual cells. We then propose a novel methodology that makes use of deconvolution techniques to extract the mean and the variability of the distribution of intake times. We find that cells, following shifts to low temperatures, have higher intake times, although, counter-intuitively, the cell-to-cell variability of these times is lower. We validate the results using a new methodology for direct estimation of mean intake times from measurements of activation times at various inducer concentrations. The results confirm that E. coli's inducer intake times from the environment are significantly higher following a shift to a sub-optimal temperature. Finally, we provide evidence that this is likely due to the emergence of additional rate-limiting steps in the intake process at low temperatures, explaining the reduced cell-to-cell variability in intake times.

Added value of autoregulation and multi-step kinetics of transcription initiation

“…two genes with similar rates of mRNA and protein production in one condition can differ widely in other conditions if the new conditions cause the relative durations of the rate-limiting steps of the two genes to differ (e.g. [13]). In recent works, it was also suggested that the effects of this phenomenon may have multi-scale effects, i.e.…”

“…Relevantly, most predictions regarding the changes in kinetics obtained from the simulations could be tested by using such already engineered circuits (e.g. [46]), by changing their original promoters for others with different initiation kinetics (strength and, in particular, relative duration of the rate-limiting steps in transcription initiation [13,18,23]). Similar tests could be performed by changing the binding affinities of TFs to the promoters, whose original values can be found in [15,92–94], as these changes are expected to also allow changes in the transcription initiation kinetics of the genes composing the circuits.…”

“…For this, instead of changing the mean transcription rate, we alter the fraction of time spent in the events prior to and after initiation of the open complex formation. Namely, we vary τ after /Δ t between 0.05 and 0.95, to cover the wide diversity of empirical values reported in [13,18]. For this, Δ t is kept constant, and k 1 and k 2 are changed according to equations (2.12)–(2.15), depending on the circuit's topology.…”

“…This is because changes in Dt are limited by biophysical constraints such as intracellular concentration of RNA polymerases, promoter affinity biophysical limitations, etc. while empirical evidence suggests that t prior /Dt can be changed from almost 0 to almost 1 [13,18]. The rates k 1 and k 2 for constitutive genes were estimated using equations (2.14) and (2.15).…”

“…in Escherichia coli, this adaptability is made possible by, among other things, fine-tuning the transcription kinetics of its genes [9]. This is enhanced by the multi-step nature of transcript initiation [10][11][12][13], whose steps can be individually or jointly controlled by promoter-specific external signals (e.g. transcription factors), global regulators such as s factors, etc.…”