Biochemical and structural studies have shown that the initiation of RNA polymerase II transcription proceeds in the following stages: assembly of the polymerase with general transcription factors and promoter DNA in a ‘closed’ preinitiation complex (PIC)1,2; unwinding of about 15 base pairs of the promoter DNA to form an ‘open’ complex3,4; scanning downstream to a transcription start site; synthesis of a short transcript, thought to be about 10 nucleotides long; and promoter escape. Here we have assembled a 32-protein, 1.5-megadalton PIC5 derived from Saccharomyces cerevisiae, and observe subsequent initiation processes in real time with optical tweezers6. Contrary to expectation, scanning driven by the transcription factor IIH7,8,9,10,11,12 involved the rapid opening of an extended transcription bubble, averaging 85 base pairs, accompanied by the synthesis of a transcript up to the entire length of the extended bubble, followed by promoter escape. PICs that failed to achieve promoter escape nevertheless formed open complexes and extended bubbles, which collapsed back to closed or open complexes, resulting in repeated futile scanning.
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We thank A. Chakraborty and B. Milic for careful reading of the manuscript, P.-J. Mattei for help with protein purification, and R. Landick and J. Gelles for discussions. This research was supported by NIH grants GM36659 and AI21144 to R.D.K. and GM57035 to S.M.B., and an NSF graduate fellowship to F.M.F.
The authors declare no competing financial interests.
Extended data figures and tables
a, PIC excluding the kinase domain (TFIIK) was assembled on SNR20* short (adjacent to the 2.7-kb downstream handle sequence) and sedimented on a glycerol gradient; fractions were analysed by SDS–PAGE. b, The results from fraction 12, annotated in detail, indicate that all PIC components were retained, confirming that the complex reconstituted fully from the component proteins. The subunit(s) of Pol II are labelled in black, TFIIF in blue, TFIIE in magenta, TFIIH in orange, TFIIA in cyan, TFIIB in red, TBP in light green, and Sub1 in dark green. TFIIK (3-subunits) was later added to the PIC.
PICs were attached to one bead via biotin-avidin linkages (yellow). To form dumbbell tethers, the other end of a small fraction of the PICs (4%) had digoxigenin linkages that could be tethered to anti-digoxigenin-coated beads (black and brown) via a 2.7-kb DNA handle. PICs not involved in tether formation served to increase the local concentration of PIC components.
a, Isolated PICs (0.1 pmol), formed on the SNR20* short promoter fragment fused to the transcription template (covering the region −62/+636), and attached to a 2.7 kb DNA handle, was combined with increasing amounts of PICs assembled on the SNR20* short promoter, but without the handle, hereafter referred to as PIC (−62/+96). These constituents were incubated with an equal volume of a 2X NTP solution (10 ml) containing 1.6 mM ATP, 1.6 mM GTP, 1.6 mM CTP, 40 mM UTP, and 0.83 mM [α-32P] UTP (2.5 μCi). The resulting transcripts were analyzed by gel electrophoresis. PICs fused to the DNA handle failed to support transcription alone (lane 1), but transcription activity was restored (red arrow) when a 4-fold (lane 2), 8-fold (lane 3), 12-fold (lane 4), or 15-fold (lane 5) excess of PIC (−62/+96) was added to the reactions. In lane 6, the reaction contains 1.5 pmol PIC (−62/+96). The 96-nt run-off transcription from PIC (−62/+96) is indicated (black arrow). A 25-fold excess of PIC (−62/+96) was used for single-molecule assays (Extended Figure. 2). b, 1.5 pmol aliquots of PIC (−62/+96) were introduced into different volumes of transcription buffer, such that assayed concentration of PIC varied from 37 nM to 4.5 nM. Transcription efficiency (run-off band, black) decreased with PIC concentration from ∼18% to just 2–3%. The low concentrations used in single-molecule assays (<1 nM) could not be assayed directly using gels, but we expect that the transcription efficiency is correspondingly low.
a, b, Just as for SNR20* short (Fig. 3), the longer promoter shows TFIIH scanning with both rNTPs (a) or dATP only (b), after which either the PIC dissociates (black arrows), or the bubble collapses to the closed (blue and green records) or open (grey line) complex and TFIIH moves again. The dashed line indicates the position of the TSS (+1).
In the absence of nucleotides in vitro, PIC complexes bound to the SNR20* long promoter produced barriers to exonuclease III digestion located ∼50 bp downstream of the TATA box (about −40 nucleotides from the TSS, black arrows). These barriers depended on the presence of TFIIH and also TFIIE, which interacts with TFIIH. After the addition of dATP, the barriers disappeared, and the bands at pause positions were intensified between positions −30 and +30 (∼60–120 bp downstream of the TATA box, bracket).
Extended Data Figure 6 The transcription initiation pathway for SNR20* long (left) and SNR20* short (right) promoters.
Left, a model for the initiation pathway on the SNR20* long promoter. States starting from the top: Pol II (beige) with attached GTFs (blue) and Ssl2 (orange) binds in its ‘closed’ form to the promoter element upstream of the TSS (arrow) on the DNA template (green and blue lines). Positions of the enzyme active site (open white circle) and TATA box (closed black square) are indicated. Unwinding by TFIIH produces an open complex (OC) that leads to bubble formation. Arrival of the open complex at the TSS owing to scanning, driven by TFIIH, leads to the formation of an extended bubble (dashed lines indicate the speculative position of single-stranded DNA). If the complex fails to recognize the TSS, it can be driven beyond it by TFIIH, resulting in a ‘fast state’ that produces no RNA but advances at roughly twice the normal rate (black box; see text). When Pol II recognizes the TSS, it begins transcription of RNA (red line), corresponding to the ITC. Formation of the ITC leads to bubble collapse, followed by the loss of GTFs and transition to the elongation complex (EC). Right, corresponding model for the initiation pathway on the SNR20* short promoter. Similar states as for SNR20* long. In this case, the open complex does not need to scan for the TSS, which is found within its DNA footprint. As a consequence, the ITC can form and begin RNA synthesis once the active site has recognized the TSS. A longer segment of RNA can thereby be produced before the transition to the elongation complex (EC).
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Fazal, F., Meng, C., Murakami, K. et al. Real-time observation of the initiation of RNA polymerase II transcription. Nature 525, 274–277 (2015). https://doi.org/10.1038/nature14882
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