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 (

Selective inhibition of CDK7 reveals high-confidence targets and new models for TFIIH function in transcription

“…The CAK complex was purchased from Millipore (CDK7/CCNH/ MAT1; CAK complex; 250 µg of 14-476M). The TFIIH purification was completed as described (Fant et al 2020).…”

Quantitative imaging of transcription in livingDrosophilaembryos reveals the impact of core promoter motifs on promoter state dynamics

“…After initiation, Pol II transcribes a short stretch of 30-80 nucleotides before pausing 7 . This step is regulated by the TFIID complex, as directly demonstrated in vitro 8 and inferred from the over-representation of TFIID-bound core promoter motifs (INR, DPE, pause button) in highly paused genes [9][10][11][12][13] . Pausing durations are highly variable among genes [14][15][16] .…”

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 °C until use. The holo- Hs TFIIH 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].…”