A comparative analysis of promoter-proximal pausing reveals kinetic and distributional dimensions of variation

Promoter-proximal pausing of RNA polymerase II is a key regulatory checkpoint in metazoan transcription. Despite extensive study of this process, quantitative methods for comparing pausing dynamics across biological contexts have been lacking. Here we introduce a model-based framework for rigorous comparative analysis of both pause-escape kinetics and pause-site distributions across genes, cell types, and species. An application to available PRO-seq datasets revealed striking differences across perturbations, and comparative analyses across cell types and species highlighted distinct patterns of variation in both pause-escape kinetics and pause-site distributions, with only weak coupling between them. Integration with chromatin and sequence features showed that lower pause-escape rates are associated with stronger promoter-proximal nucleosome occupancy, whereas changes in pause-site dispersion are associated with sequence features such as GC skew. Together, these results establish a quantitative framework for comparative analysis of promoter-proximal pausing and reveal kinetic and distributional dimensions of pausing variation across biological contexts.