Many proteins, including RNA polymerase II (Pol II) and a variety of transcription factors, are involved in the transcription process by assembling into protein enriched phases through complex spatial mechanisms. To better understand the molecular interactions underlying the phase separation during transcription, we investigated the clustering behavior of Pol II and Cyclin T1, a subunit of the positive transcription elongation factor b (PTEF b). Earlier studies show that condensates form between two specific domains of Pol II and cyclin T1, which are the C-terminal domain (CTD) and the histidine rich domain (HRD), respectively. These two domains are intrinsically disordered, meaning they have no definite structures, and have a higher flexibility, which allows them to form multivalent intermolecular interactions that may lead to cluster formation and phase separation. We performed molecular dynamics (MD) simulations of concentrated systems that have HRD of cyclin T1 and a 2 heptapeptide model of CTD of Pol II. With only HRD systems at different concentrations, we found small amounts of interactions between the segments, without any clustering pattern. This was expected as HRD is highly electropositive, which creates a repulsion between the peptides. However, when we added CTD to the HRD system, we saw a large increase in clustering with the non-phosphorylated CTD and an even larger increase with the phosphorylated CTD. We propose that the increase in clustering with the presence of CTD has a functional importance, such that the increased concentration of cyclin t1 and CTD in condensates may promote binding of Pol II with the PTEFb complex, which further promotes phosphorylation of Pol II by the CDK9 domain of PTEFb.
Oakden et al. (Sun,) studied this question.