Model-based characterization of the equilibrium dynamics of transcription initiation and promoter-proximal pausing in human cells

In metazoans, both transcription initiation and the escape of RNA polymerase (RNAP) from promoter-proximal pausing are key rate-limiting steps in gene expression. These processes play out at physically proximal sites on the DNA template and appear to influence one another through steric interactions, leading to a complex dynamic equilibrium in RNAP occupancy of the ~100 bp immediately downstream of the transcription start site. In this article, we examine the dynamics of these processes using a combination of statistical modeling, simulation, and analysis of real nascent RNA sequencing data. We develop a simple probabilistic model that jointly describes the kinetics of transcription initiation, pause-escape, and elongation, and the generation of nascent RNA sequencing read counts under steady-state conditions. We then extend this initial model to allow for variability across cells in promoter-proximal pause site locations and steric hindrance of transcription initiation from paused RNAPs. In an extensive series of simulations over a broad range of parameters, we show that this model enables accurate estimation of initiation and pause-escape rates even in the presence of collisions between RNAPs and variable elongation rates. Furthermore, we show by simulation and analysis of data for human cell lines that pause-escape is often more strongly rate-limiting than conventional "pausing indices" would suggest, that occupancy of the pause site is elevated at many genes, and that steric hindrance of initiation can lead to a pronounced reduction in apparent initiation rates. Our modeling framework is generally applicable for all types of nascent RNA sequencing data and can be applied to a variety of inference problems.