Abstract Background In 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. Results We 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. Conclusions The 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.