Abstract
RNA polymerase II (RNAPII) transcription is governed by the pre-initiation complex (PIC), which contains TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH, RNAPII, and Mediator. After initiation, RNAPII enzymes pause after transcribing less than 100 bases; precisely how RNAPII pausing is enforced and regulated remains unclear. To address specific mechanistic questions, we reconstituted human RNAPII promoter-proximal pausing in vitro, entirely with purified factors (no extracts). As expected, NELF and DSIF increased pausing, and P-TEFb promoted pause release. Unexpectedly, the PIC alone was sufficient to reconstitute pausing, suggesting RNAPII pausing is an inherent PIC function. In agreement, pausing was lost upon replacement of the TFIID complex with TATA-binding protein (TBP), and PRO-seq experiments revealed widespread disruption of RNAPII pausing upon acute depletion (t = 60 min) of TFIID subunits in human or Drosophila cells. These results establish a TFIID requirement for RNAPII pausing and suggest pause regulatory factors may function directly or indirectly through TFIID.
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•Reconstitution of RNAPII pausing in vitro, with purified factors (no extracts)•Human PIC alone is sufficient to establish RNAPII pausing•TFIID is required for RNAPII promoter-proximal pausing•Rapid TFIID depletion induces RNAPII pause release genome-wide
Fant and co-workers use biochemical reconstitution to discover that the general transcription factor TFIID is required to establish RNAPII promoter-proximal pausing; canonical regulatory factors NELF and DSIF enhance pausing but are not required. Follow-up experiments in human and Drosophila cells show TFIID is a conserved regulator of RNAPII pausing.