TFIID Enables RNA Polymerase II Promoter-Proximal Pausing

Abstract

TFIIH, Mediator, and RNAPII (Figure S1). Experiments were completed with the native human HSP70 promoter (HSPA1B gene), because others have shown that it is a quintessential model for promoter-proximal RNAPII pausing (Core et al., 2012). Because chromatin per se does not appear to be an essential regulator of RNAPII pausing in Drosophila or mammalian cells Lai and Pugh, 2017;Li et al., 2013), the in vitro transcription assays were completed on naked DNA templates (also see below).Using purified PIC factors, primer extension assays established that transcription initiation occurred at the annotated HSPA1B start site in vitro (Figure S2A), as expected. An overview of the transcription assay is shown in Figure 1A, which was based in part upon in vitro pausing assays with nuclear extracts (Marshall and Price, 1992;Qiu and Gilmour, 2017;Renner et al., 2001). Following PIC assembly, transcription was initiated by adding ATP, GTP, and UTP at physiologically relevant concentrations, with a low concentration of CTP, primarily 32 P-CTP. After one minute, reactions were chased with a physiologically relevant concentration of cold CTP and transcription was allowed to proceed for an additional nine minutes. These "pulse-chase" assays allow better detection of short (potentially paused) transcripts, which otherwise would be drowned out by elongated transcripts that invariably possess more incorporated 32 P-C bases. By directly labeling all transcripts with 32 P-CTP, the method is highly sensitive and allowed detection of transcripts of varied lengths; furthermore, the 32 P-CTP pulse-chase protocol ensured that 32 P-labeled transcripts resulted almost exclusively from single-round transcription (see Methods). Control experiments confirmed that transcripts detected were driven by the HSP70 promoter (e.g. not any contaminating nucleic acid) and that transcription was dependent on added PIC factors, as expected (Figure S2B).A variety of methods have established that RNAPII pauses after transcribing 20-80 bases in Drosophila and mammalian cells (

A unified view of the sequence and functional organization of the human RNA polymerase II promoter

“…While this paper was in review, Fant et al. ( 60 ) reported that TFIID is essential for pausing downstream of both Drosophila and human promoters. Given that pausing occurs at nearly all Pol II promoters ( 18–20 ), this finding independently supports the idea that Pol II promoters are centered on TFIID binding sites.…”

Negative elongation factor regulates muscle progenitor expansion for efficient myofiber repair and stem cell pool repopulation

“…Although pause release is subsequently induced through the recruitment of P-TEFb in response to signals from the environment, the paused RNAPII is prone to arrest or premature termination (Adelman et al, 2005;Elrod et al, 2019). To prevent this, the negative elongation factor (NELF) complex is recruited to stabilize paused RNAPII (Henriques et al, 2013) through impeding RNAPII interactions with positive mediators of transcription elongation (Vos et al, 2018) and facilitating transcription re-initiation (Boettiger and Levine, 2009;Fant et al, 2020;Gilchrist and Adelman, 2010). As such, the loss of NELF leads to reduced RNAPII accumulation and transcription termination within the proximal-promoter region of its target genes (Core and Adelman, 2019).…”

The role of XPB/Ssl2 dsDNA translocation processivity in transcription-start-site scanning

“…The elution with TFIIH core complex (confirmed by SDS-PAGE gel and 420 nm absorption peak) was stored at -80 o C until use. The holo-HsTFIIH purification was completed as described [38].…”

“…The S. cerevisiae Pol II RNA polymerase and the other general transcription factors (TBP, TFIIB, TFIIF, and TFIIE) were purified as previously described [39]. The human factors (Pol II, TBP, TFIIB, TFIIF, TFIIE, and TFIIH) were purified as described [38].…”