Biomolecular condensates play a crucial role in various biological processes. In the context of transcription, these condensates help concentrate key regulatory proteins at super-enhancers, which are responsible for activating genes that define cell identity. They may also help explain how enhancers influence gene expression over large genomic distances. However, due to their diffraction limited size (<300 nm), studying their physical properties and testing the phase separation model for their formation remains challenging. We use single-particle tracking to investigate the behavior of individual RNA polymerase II (Pol II) molecules within transcription condensates in live mouse embryonic stem cells. We compare the movement of Pol II within these condensates to its dynamics in the surrounding nucleoplasm. Our analysis identifies a DNA-bound population and two distinct mobile states of Pol II. Surprisingly, we find that nearly all Pol II molecules inside transcription condensates are immobile. This observation is supported by the fact that Pol II within condensates is highly phosphorylated at serine 5, indicating that most Pol II in these regions is engaged in transcription initiation and bound to promoter-proximal DNA. When transcription initiation is inhibited, Pol II becomes more mobile both within the nucleus and in the condensates. Interestingly, while Pol II is progressively lost from condensates under these conditions, the Mediator complex remains. We propose that the local accumulation of Pol II in transcription condensates is driven by its efficient recruitment to DNA for transcription initiation. In contrast to what has been observed in vitro, the C-terminal domain (CTD) of Pol II shows only weak partitioning into condensates in live cells. Overall, our findings suggest that Pol II is enriched within transcription condensates primarily through chromatin interactions and transcription initiation rather than through passive, CTD-mediated partitioning alone.
Budhathoki et al. (Sun,) studied this question.