In vivo kinetics of transcription initiation of the lar promoter in Escherichia coli. Evidence for a sequential mechanism with two rate-limiting steps

Abstract

BackgroundIn Escherichia coli the mean and cell-to-cell diversity in RNA numbers of different genes vary widely. This is likely due to different kinetics of transcription initiation, a complex process with multiple rate-limiting steps that affect RNA production.ResultsWe measured the in vivo kinetics of production of individual RNA molecules under the control of the lar promoter in E. coli. From the analysis of the distributions of intervals between transcription events in the regimes of weak and medium induction, we find that the process of transcription initiation of this promoter involves a sequential mechanism with two main rate-limiting steps, each lasting hundreds of seconds. Both steps become faster with increasing induction by IPTG and Arabinose.ConclusionsThe two rate-limiting steps in initiation are found to be important regulators of the dynamics of RNA production under the control of the lar promoter in the regimes of weak and medium induction. Variability in the intervals between consecutive RNA productions is much lower than if there was only one rate-limiting step with a duration following an exponential distribution. The methodology proposed here to analyze the in vivo dynamics of transcription may be applicable at a genome-wide scale and provide valuable insight into the dynamics of prokaryotic genetic networks.

Regulation of mean and noise of the in vivo kinetics of transcription under the control of the lac/ara‐1 promoter

“…Two studies reported in vivo measurements of the kinetics of P lac/ara-1 [21,22]. In these, the RNA is visible shortly after transcription occurs by tagging them with MS2d-GFP proteins [23].…”

Effects of multimerization on the temporal variability of protein complex abundance

“…In our implementation, each time this reaction occurs, a value of τ S is drawn from a gamma distribution with mean of k M -1 and coefficient of variation (variance over the mean) of ${\alpha }_M^{MathClass-bin-1MathClass-bin/2}$. With proper parameter values, the gamma distribution well approximates recent live cell measurements of intervals between productions of consecutive RNAs in E. coli [13,20]. We fitted the measurements of time intervals in [20] with the three-exponential model proposed in that work, and with a gamma distribution.…”

“…This approximation assumes that the elongation time is negligible, which relies on observation that the durations of the closed and the open complex formations (in the order of 10 3 s) [1,2,13,20] are much longer than elongation (in the order of 10 1 or 10 2 s) [21,22]. Moreover, in prokaryotes, translation is coupled to transcription [23], and can initiate as soon as the ribosome binding site region (RBS) of the RNA is formed (Shine-Dalgarno sequence) [24].…”

“…The RNA production rate of a gene is mainly controlled during the process of transcription initiation, at the promoter region (see [1] for a review). Recent in vivo measurements of the intervals between the production of individual transcripts [13,14] suggest that, under normal growth conditions, there are two to three significant rate-limiting steps at the initiation stage that, aside from determining the mean rate of production, also determine the degree of noise in the process of RNA production. In prokaryotes, these observations relate directly to protein copy numbers, which tend to follow closely those of RNA [15].…”

“…To account for the stochasticity and the rate limiting steps of the underlying steps in the process of gene expression, we use the delayed stochastic modeling strategy [16] to drive the dynamics of the models, as it allows the use of non-Markovian dynamics to model the non-instantaneous processes underlying transcription and translation [17]. The parameters used in the models are extracted from live, single-cell, single-molecule measurements [6,13,14]. …”