The Role of the Transcription Bubble and TFIIB in Promoter Clearance by RNA Polymerase II  Mahadeb Pal, Alfred S. Ponticelli, Donal S. Luse  Molecular Cell  Volume 19, Issue 1, Pages 101-110 (July 2005) DOI: 10.1016/j.molcel.2005.05.024 Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 1 Relative Stabilities of Transcription Complexes Assembled on Variants of the Ad ML Promoter (A) The nontemplate strand sequences of the promoters used are shown; boldface italics indicate the segment transcribed without GTP. Distances between the TATA box and the +1 site were counted from the boldface A. The underlined bases in the 6g and 8g series were deleted in the 2D promoters; bases in lower case were inserted to build the 2I and 4I promoters. The right-hand columns (% stable) show the percent of transcription complexes originally stalled at the G stop that remained template bound after a 3 min (NE) or 1 min (PF) incubation at 30°C followed by chase to the initial A stop. PICs were assembled in nuclear extracts (NE) or with purified factors (PF). The values shown are the averages of two or three experiments. Individual measurements did not vary more than 5% from the averages. (B) (Left) PICs were assembled on the 6g promoters with nuclear extracts, followed by transcription to the G stop, washing, and incubation at 30°C for 3 min. (Right) PICs were assembled on the 8g promoters with purified pol II and transcription factors, followed by transcription to the G stop, washing, and incubation for 1 min at 30°C. For both panels, half of the initial G-less reaction was removed (T), while the other half was chased to the A stop. Chase reactions were separated into bead bound (B) and unbound (U) fractions. Percent stable was calculated as in (A). Molecular Cell 2005 19, 101-110DOI: (10.1016/j.molcel.2005.05.024) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 2 Determination of Transcription Bubble Dimensions for Complexes on the 8g and 9g Promoters (A) (Left-hand panels) Transcription complexes, initiated as indicated, were advanced to the G stop on the 8g templates and treated with KMnO4. Positions of reactive thymines (on the right in each panel) were determined by comigration with G+A sequence ladders (not shown). Complexes processed prior to transcript initiation are marked PIC; direct reaction of KMnO4 with template DNA alone was done in the DNA lane. The gray bar adjacent to the 8g2I panel indicates a region of reduced KMnO4 reactivity. The asterisk at position −6 of the 8g2D panel does not correspond to a T residue, but reactivity at this location was reproducibly observed. (Right-hand diagram) The ellipses indicate the minimum extent of the unpaired regions on the nontemplate strands. Unpaired regions were also determined for complexes halted at +7 on other 8G series promoter variants with additional T residues between positions −6 and −10 (data not shown). These assays revealed weak reactivity on the 8g2D promoter at −7 (light gray bracket) and stronger reactivity on the 8gW promoter at −9 (gray bracket). The bubble size figures in the right-hand column reflect these more extended unpaired regions. The gray bar within the 8g2I ellipse indicates reduced KMnO4 reactivity. (B) (Left-hand panels) As in (A), except with 9g promoters; transcription was initiated only with ApC. (Right-hand diagram) As in (A), except that in this case no additional promoters were tested to map the upstream bubble edge. Thus, the bubble sizes are designated as minimum sizes. Molecular Cell 2005 19, 101-110DOI: (10.1016/j.molcel.2005.05.024) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 3 Determination of Transcription Bubble Dimensions for Complexes on the 6g Promoters (Upper panels) Transcription complexes, initiated as indicated, were advanced to the G stop on the 6g templates and treated with KMnO4. Positions of reactive thymines (on the right of each panel) were determined by comigration with G+A sequence ladders (not shown). Reactions processed prior to transcription are marked PIC. (Lower diagram) The ellipses indicate the minimum extent of the unpaired regions on the nontemplate strands. Unpaired regions were also determined for transcription complexes halted at +5 on other 6g series promoter variants with additional T residues between positions −6 and −10 (data not shown). These assays revealed weak reactivity on the 6g2D promoter at −7 (light gray bracket) and stronger reactivity on the 6gW promoter at −9 (gray bracket). The bubble size figures in the right hand column reflect these more extended unpaired regions. Molecular Cell 2005 19, 101-110DOI: (10.1016/j.molcel.2005.05.024) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 4 Dependence of Transcript Elongation on dATP for the 8g Promoters PICs assembled with purified factors were advanced to the G stops of the indicated promoters after initiation with CpA. After washing, aliquots of the stalled complexes were chased with 200 μM each of CTP, UTP, and GTP, ±40 μM dATP, for 2 min at 30°C. Fold-stimulation by dATP was calculated by comparing the intensities of 23 nt runoff bands obtained with and without dATP. The status of the upstream segment of the transcription bubble is noted for each complex (see Figure 2). Molecular Cell 2005 19, 101-110DOI: (10.1016/j.molcel.2005.05.024) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 5 The Relationship among Bubble Size, TATA to G Stop Spacing, and Dependence on the TFIIH Helicase The stimulation of transcript elongation by dATP was determined for the 6g, 8g, and 9g series of transcription complexes, as well as for complexes having wild-type TATA to +1 spacing and G stops at +11, +13, or +15. The dATP stimulation values, determined as described in Figure 4, are averages of at least three independent experiments; individual measurements did not deviate more than 5% from these averages. The data points are open, hatched, or solid to indicate the status (open, closing, or closed) of the upstream segment of the transcription bubble in the complex in question. Stimulation values are plotted versus both the spacing of TATA to the G stop as well as the bubble size for each complex (see Figures 2 and 3). The longest bubbles in our complexes were 18 bases (circle); for complexes in which larger bubbles would be predicted, the upstream segment of the bubble had reannealed. We did not measure bubbles for the 11g, 13g, and 15g complexes but simply assumed that the upstream segment had closed. Molecular Cell 2005 19, 101-110DOI: (10.1016/j.molcel.2005.05.024) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 6 Pausing by Pol II from +7 to +9 during Transcription of a Bubble Template Depends on TFIIB and Is Diminished with a TFIIB Variant in the B Finger Domain (A) Preinitiation complexes were assembled on bead-attached double-stranded (ds, lanes 1 and 2) or premelted (−9/−1) templates with all the general transcription factors (GTFs) present or one of the GTFs absent (lanes 3–14). Transcription was initiated with CpA, 20 μM UTP, and 1 μM [α-32P]CTP with or without 20 μM dATP for 5 min at 30°C, followed by an additional 2 min incubation with 20 μM nonlabeled CTP. Transcripts in the bead bound fractions were processed and analyzed as described in Experimental Procedures. (B) PICs were assembled on the indicated templates with all GTFs including either wild-type TFIIB (lanes 1–4) or the R66L variant of TFIIB (lanes 5–8). Transcription was performed as in (A). Molecular Cell 2005 19, 101-110DOI: (10.1016/j.molcel.2005.05.024) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 7 A Model of the Promoter Clearance Process Several of the important interactions during the earliest stage of transcript elongation by pol II are shown in schematic form. As the transcript is elongated, the transcription bubble stretches and complex stability decreases. Once bubble collapse has occurred, stability is recovered. TFIIH is shown as a hatched shape surrounding the RNA polymerase, contacting DNA both upstream and downstream of the complex, as suggested by Douziech et al. (2000) (but see also Kim et al., 2000). Bubble collapse requires the synthesis of at least a 7 nt RNA. We suggest that the presence of RNA of this length forces rearrangement of portions of the transcription complex (cross-hatched shape); as noted in the Discussion, this rearrangement probably involves TFIIB. Molecular Cell 2005 19, 101-110DOI: (10.1016/j.molcel.2005.05.024) Copyright © 2005 Elsevier Inc. Terms and Conditions