Rate-limiting steps in transcription dictate sensitivity to variability in cellular components

Abstract

Cell-to-cell variability in cellular components generates cell-to-cell diversity in RNA and protein production dynamics. As these components are inherited, this should also cause lineage-to-lineage variability in these dynamics. We conjectured that these effects on transcription are promoter initiation kinetics dependent. To test this, first we used stochastic models to predict that variability in the numbers of molecules involved in upstream processes, such as the intake of inducers from the environment, acts only as a transient source of variability in RNA production numbers, while variability in the numbers of a molecular species controlling transcription of an active promoter acts as a constant source. Next, from single-cell, single-RNA level time-lapse microscopy of independent lineages of Escherichia coli cells, we demonstrate the existence of lineage-to-lineage variability in gene activation times and mean RNA production rates, and that these variabilities differ between promoters and inducers used. Finally, we provide evidence that this can be explained by differences in the kinetics of the rate-limiting steps in transcription between promoters and induction schemes. We conclude that cell-to-cell and consequent lineage-to-lineage variability in RNA and protein numbers are both promoter sequence-dependent and subject to regulation.

The route to transcription initiation determines the mode of transcriptional bursting in E. coli

“…A fuller understanding of the sources of transcriptional noise and bursting in bacteria requires an appreciation of the biochemical complexity of transcription regulation to avoid oversimplified views of the events that lead to open complex formation during transcription initiation. The impact of transcription initiation on noise and bursting has been acknowledged 57,58 . Yet, the contribution of sigma factors-whose role is to facilitate transcription initiationhas so far been overlooked.…”

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

“…Previous studies showed that the sensitivity of a promoter's activity to TFs is affected by τ prior /Δ t , when TFs do not affect identically the kinetics of the rate-limiting steps in transcription initiation [18,23] (figure 1 f ). For example, consider two TFs with similar repressing capabilities, with one being able to double the mean duration of the first rate-limiting step, while the other can double the duration of the second rate-limiting step.…”

“…affinity of activators to the binding site0 molecule −1 [46]min. affinity of repressors to the binding site0.01 molecule −1 [46] K I half-maximal concentration of inducer for activator2.5% (w/v)[46]half-maximal concentration of inducer for repressor0.035 mM[46] k 1 binding affinity of the RNAP to the promoter+[12] k −1 unbinding affinity of the RNAP from the promoter60 min −1 [12] k 2 effective rate of events after transcription initiation until mRNA synthesis+[12] k p rate of translation27 protein min −1 [63–65] d mRNA rate of mRNA degradation0.12 min −1 [60,66] d protein rate of protein/activator/repressor degradation0.0231 min −1 [67]RNAPnumber of free RNA polymerases1000[68–70] I inducer concentration for activators[0–2.5] % (w/v)[23,42,47]inducer concentration for repressor[0–1] mM[23,42,47]Δ t avg. duration of transcription intervals[1–20] min[12,…”

“…Relevantly, the transcription dynamics is sequence dependent because e.g. the promoter sequence affects the kinetics of the rate-limiting steps in the initiation, altering the mean and cell-to-cell variability in RNA, and thus, protein numbers [15,18,23–25].…”

“…Based on past knowledge (see e.g. [23]) we predict that the effects of autoregulation depend not only on the mode and strength of this regulation but also on the dynamics of transcription initiation of the gene under regulation.…”