Ample evidence demonstrates that many intrinsically disordered protein regions (IDRs) have the ability to accumulate in biomolecular condensates via a phase separation process. In vitro reconstitutions allow for the construction of detailed phase diagrams. It is much more challenging to determine if the phase separating properties are of practical importance in the vastly more complex intracellular environment. Here, we use single molecule tracking to investigate how constituent proteins of transcription condensates and their IDRs explore endogenous condensates in living cells. We find that RNA Pol II enrichment in condensates is largely due to DNA binding rather than a phase separation process. The intrinsically disordered C-terminal domain of the largest Pol II subunit Rpb1 facilitates Pol II recruitment to condensates but by itself partitions only weakly into condensates consisting of Mediator and Brd4. Accordingly, removal of Pol II from condensates by acute degradation does not affect partitioning of the other constituents. High levels of Serine 5-phosphorylation reflect abundance of initiating and transcribing Pol II in condensates. Enrichment of promoter epigenetic marks in condensates revealed by multicolor 3D super-resolution microscopy provides further support for a model in which mobile Pol II moves freely into and out of the condensate phase, and is retained by efficient loading onto promoter DNA. IDR interactions may facilitate this process but are not sufficient to explain Pol II partitioning into condensates. In ongoing work, we apply the experimental framework to characterize the behavior of other constituent fractions and their IDRs in multicomponent transcription condensates.
Jan-Hendrik Spille (2026) studied this question.