Real-time visualization of reconstituted transcription reveals RNA polymerase II activation mechanisms at single promoters DOI Creative Commons
Megan Palacio, Dylan J. Taatjes

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2025, Volume and Issue: unknown

Published: Jan. 6, 2025

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 Mediator contain intrinsically-disordered regions (IDRs) 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 at hundreds promoters simultaneously. We show rapid activation IDR-dependent, without condensate formation. For example, MED1-IDR can functionally replace native TF, activating with similar (not identical) kinetics; however, squelches as condensate, activates single-protein. cooperatively activate bursting re-initiation surprisingly, drive TF-promoter recruitment, TF-DNA binding. Collectively, RIFT addressed questions largely intractable cell-based methods, yielding mechanistic insights about IDRs, enhancer-promoter communication, that complement live-cell imaging data.

Language: Английский

Real-time visualization of reconstituted transcription reveals RNA polymerase II activation mechanisms at single promoters DOI Creative Commons
Megan Palacio, Dylan J. Taatjes

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2025, Volume and Issue: unknown

Published: Jan. 6, 2025

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 Mediator contain intrinsically-disordered regions (IDRs) 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 at hundreds promoters simultaneously. We show rapid activation IDR-dependent, without condensate formation. For example, MED1-IDR can functionally replace native TF, activating with similar (not identical) kinetics; however, squelches as condensate, activates single-protein. cooperatively activate bursting re-initiation surprisingly, drive TF-promoter recruitment, TF-DNA binding. Collectively, RIFT addressed questions largely intractable cell-based methods, yielding mechanistic insights about IDRs, enhancer-promoter communication, that complement live-cell imaging data.

Language: Английский

Citations

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