Real-time visualization of reconstituted transcription reveals RNA polymerase II activation mechanisms at single promoters.

RNA polymerase II (RNAPII) is regulated by sequence-specific transcription factors (TFs) and the pre-initiation complex (PIC): TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH, Mediator. TFs and Mediator contain intrinsically-disordered regions (IDRs) and form phase-separated condensates, but how IDRs control RNAPII function remains poorly understood. Using purified PIC factors, we developed a Real-time In-vitro Fluorescence Transcription assay (RIFT) for second-by-second visualization of RNAPII transcription at hundreds of promoters simultaneously. We show rapid RNAPII activation is IDR-dependent, without condensate formation. For example, the MED1-IDR can functionally replace a native TF, activating RNAPII with similar (not identical) kinetics; however, MED1-IDR squelches transcription as a condensate, but activates as a single-protein. TFs and Mediator cooperatively activate RNAPII bursting and re-initiation and surprisingly, Mediator can drive TF-promoter recruitment, without TF-DNA binding. Collectively, RIFT addressed questions largely intractable with cell-based methods, yielding mechanistic insights about IDRs, condensates, enhancer-promoter communication, and RNAPII bursting that complement live-cell imaging data.