The Role of the Transcription Bubble and TFIIB in Promoter Clearance by RNA Polymerase II

Molecular Cell, Vol. 19, 101–110, July 1, 2005, Copyright ©2005 by Elsevier Inc. DOI 10.1016/j.molcel.2005.05.024

The Role of the Transcription Bubble and TFIIB in Promoter Clearance by RNA Polymerase II Mahadeb Pal,1 Alfred S. Ponticelli,2 and Donal S. Luse1,* 1 Department of Molecular Genetics Lerner Research Institute Cleveland Clinic Foundation Cleveland, Ohio 44195 2 Department of Biochemistry School of Medicine and Biomedical Sciences State University of New York at Buffalo Buffalo, New York 14214

Summary We have studied promoter clearance at a series of RNA polymerase II promoters with varying spacing of the TATA box and start site. We find that regardless of promoter spacing, the upstream edge of the transcription bubble forms 20 bp from TATA. The bubble expands downstream until 18 bases are unwound and the RNA is at least 7 nt long, at which point the upstream w8 bases of the bubble abruptly reanneal (bubble collapse). If either bubble size or transcript length is insufficient, bubble collapse cannot occur. Bubble collapse coincides with the end of the requirement for the TFIIH helicase for efficient transcript elongation. We also provide evidence that bubble collapse suppresses pausing at +7 to +9 caused by the presence of the B finger segment of TFIIB within the complex. Our results indicate that bubble collapse defines the RNA polymerase II promoter clearance transition. Introduction In order to understand the controlled expression of genes, it is essential to uncover the fundamental mechanisms through which RNA polymerase II (pol II) functions. Biochemical studies and the recent determination of high-resolution structures for both free poI II (Armache et al., 2003; Bushnell and Kornberg, 2003) and a minimal transcript elongation complex (Westover et al., 2004; Kettenberger et al., 2004) have provided considerable insight into the basic process of extending the RNA chain. However, while the components necessary to achieve specific initiation have been known for some time, the transition from the initiation complex to the transcript elongation complex is not well understood. This transition must involve the disruption of numerous protein-protein contacts of pol II with initiation factors and promoter elements (Buratowski, 2000; Asturias, 2004; Bushnell et al., 2004). The point at which the polymerase has broken its initiationspecific contacts and becomes committed to transcript elongation is often referred to as promoter clearance. During the formation of roughly the initial 15 bonds, the pol II transcription complex acquires several characteristics of the mature transcript elongation complex *Correspondence: [email protected]

(recently reviewed by Dvir, 2002; Dvir et al., 2001). The length of the RNA-DNA hybrid in the elongation complex is w8 bp (Westover et al., 2004; but see also Kettenberger et al., 2004), which is also the minimum length necessary for stability in transcription complexes assembled directly from pol II, template DNA, and RNA oligonucleotides (Kireeva et al., 2000). The size of the unwound template segment, or transcription bubble, in the pol II elongation complex is not known precisely, but studies with E. coli RNA polymerase suggest that the bubble should have a relatively constant length during chain elongation (Revyakin et al., 2004). However, downstream propagation of the pol II transcription bubble is initially discontinuous (Holstege et al., 1997). The upstream edge of the bubble in the preinitiation open complex is maintained during the initial phases of transcript elongation, resulting in a bubble which stretches to w17 bases after eight bonds have been made. During formation of the next two bonds, the upstream w9 bases of the bubble reanneal, leaving only 10 bases unwound (Holstege et al., 1997). We refer to this abrupt reclosure of the upstream segment of the pol II transcription bubble as bubble collapse. The earliest phase of transcript elongation is inefficient unless the XPB helicase of TFIIH is active (Dvir et al., 1997; Liu et al., 2001), and transcription at this stage can be blocked by the addition of the helicase inhibitor γ-thio ATP (Dvir et al., 1996). Elongation becomes independent of the TFIIH helicase and resistant to γ-thio ATP at about +15 (Dvir et al., 1997; Kumar et al., 1998). Recent structural studies with complexes of TFIIB and pol II (Bushnell et al., 2004; Chen and Hahn, 2004) have demonstrated that the N-terminal segment of TFIIB reaches into pol II through the presumed RNA exit channel and extends to the catalytic center of the polymerase. It is predicted that the B finger domain of TFIIB will clash with the advancing RNA once the transcript is more than 5–6 nt (Bushnell et al., 2004). While all of the events just mentioned are features of the initiation-elongation transition, their mechanistic relationship to each other and to promoter clearance has not been established. We determined transcription complex stability and helicase dependence as a function of transcription bubble size for sets of pol II transcription complexes. We find that stability decreases with increasing bubble size up to the point of bubble collapse, after which stability is restored. We also find that complexes undergo the bubble collapse transition only when both a 17–18 base bubble and a minimum of 7 bp DNA-RNA hybrid have been made. The completion of bubble collapse marks independence from TFIIH helicase activity. Finally, we report that bubble collapse suppresses pausing at +7 to +9 that is associated with the presence of TFIIB in the transcription complex. We conclude that bubble collapse defines the pol II promoter clearance transition. Results The transcript from the adenovirus major late (Ad ML) promoter begins with the short repetitive sequence

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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).

ACUCUCU. We have shown that if pol II pauses after the second or third CU repeat, the polymerase can shift the RNA-DNA hybrid upstream and continue RNA synthesis after re-pairing the transcript with the template (Pal and Luse, 2002). The transcription complex must be somewhat unstable for hybrid disassociation and reformation to occur. Thus, the extent of transcript slippage should be a function primarily of the length and sequence of the RNA at the point of slippage. However, a remarkable feature of the slippage reaction is its strong dependence on the exact point at which RNA synthesis initiates. To summarize the observations from our earlier work, forcing initiation upstream of the normal start site with the appropriate dinucleotide primers resulted in little or no slippage, while forcing initiation downstream of the normal location resulted in greater slippage compared to the normal start site (Pal and Luse, 2002). In light of our earlier results, it seemed likely to us that the stability of the newly initiated transcription complex depends in a completely unanticipated way on the position of the transcript initiation site within the overall promoter. Since the assembly of the pol II preinitiation complex at the Ad ML promoter is anchored by the interaction of TBP with the TATA box (Hahn, 2004), we hypothesized that deviations from the wild-type spacing between TATA and the start site could result in changes in internal stress within the transcription complex, and thus in alterations in complex stability. To test this idea, we generated a series of variants of the Ad ML promoter. As diagramed in Figure 1, each promoter

set features a G stop at a common location. Within each set, we altered the distance between TATA and +1 by adding or subtracting bases at position −8, a location which does not overlap with known promoter consensus elements. Given that the maximum length of the RNA-DNA hybrid is expected to be 8–9 bp (Gnatt et al., 2001; Westover et al., 2004), we placed the G stops so that we could pause pol II just before, at, or just beyond the point at which the hybrid reaches its mature length. Each promoter set allows us to compare the relative stabilities of transcription complexes with identical transcripts differing only in the spacing of their TATA boxes and transcription start sites.

Stabilities of Transcription Complexes Stalled after Synthesis of Short RNAs Depend on the Spacing between their TATA Boxes and Transcription Start Sites Preinitiation complexes (PICs) were assembled by incubating bead-attached promoter DNA fragments (Figure 1A) with HeLa nuclear extracts or with purified pol II and transcription factors. Complexes were advanced to their respective G stops, washed with transcription buffer, incubated at 30°C, and then chased with saturating concentrations of GTP, UTP, and CTP along with dATP as energy source. The 30°C incubations were performed for 1 min (purified factor complexes) or 3 min (nuclear extract complexes); in our hands, complexes assembled with purified factors were consistently less stable. The chase reactions were separated into bead

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bound (B) and unbound (U) fractions. We defined stability as the fraction of transcripts which remained with the template bound complex. Results with complexes assembled on the 6g series of promoters using nuclear extracts are shown in Figure 1B, left panel. On these promoters, stability decreased with increasing TATA to +1 spacing. Similar results were obtained with complexes assembled with purified factors (values in Figure 1A). Transcripts longer than 5 nt in Figure 1B, left panel, or longer than 7 nt in Figure 1B, right panel, result from transcript slippage and not from read-through of the G stop. A detailed analysis of this point was presented earlier (Pal and Luse, 2002). Transcript slippage assays on the 6g promoters (slippage data not shown) indicated that slippage varies inversely with the stability values reported in Figure 1, as we had expected. The relative stabilities of 6g complexes suggested a simple relationship: the greater the distance from TATA to transcription start, the less stable the complex. However, similar stability assays with complexes on the 8g and the 9g series of promoters did not give results consistent with this model. For the 8g series, stability was higher for 8g2D complexes than 8gW complexes, but 8g2I and 8g4I complexes showed higher stability than 8gW. An example of these tests, using complexes assembled with purified factors, is shown in Figure 1B, right panel. Note that 8gW complexes are significantly less stable than 6gW complexes (with the identical TATA to +1 spacing), even though the 6gW complexes have a shorter RNA-DNA hybrid. The stability of complexes on the 9g series of templates also increased as the TATA to +1 spacing increased (Figure 1A). The Stabilities of Paused Complexes Decline until the Upstream Segment of the Transcription Bubble Reanneals We were initially surprised that the stabilities of the paused transcription complexes did not correlate in a simple way with either transcript length or the distance between TATA and transcription start. We then realized that the behavior of the transcription bubble during the earliest stage of transcript elongation might provide an explanation. Holstege et al. (1997) demonstrated that the upstream edge of the melted region at the Ad ML preinitiation complex is located at −9. As transcription proceeds, this edge remains fixed until an 8-mer RNA has been made, at which point the bubble is 17 bases long. When the RNA grows to 9 nt, the entire upstream segment of the bubble begins to reanneal (bubble collapse). Bubble collapse is essentially complete when the RNA is extended by one more nucleotide. As a working hypothesis, we supposed that increasing the size of the transcription bubble might increase strain within the complex and thus decrease stability. In this model, the stability minimum would occur just before bubble collapse begins. We further supposed that the upstream edge of the bubble would be fixed relative to the TATA box regardless of the spacing between TATA and transcription start, as suggested by the results of Giardina and Lis (1993). One would then expect that the point of minimal stability in the early elongation process could be altered, in a series of com-

plexes with identical transcript length, by altering the TATA to +1 spacing. This model is consistent with the results we report in Figure 1. Since our model predicts that bubble collapse is directly correlated with a recovery of transcription complex stability, it was necessary to determine the transcription bubble dimensions for our various stalled transcription complexes. We measured these parameters using the KMnO4 sensitivity assay (Holstege et al., 1997). Results with complexes on the 8g series of templates are shown in Figure 2. For 8gW complexes, reactive thymine residues extended from position −8 to +7 irrespective of the initiating nucleotide. The upstream edge of the transcription bubble in an initiating complex on the Ad ML promoter was previously reported to be located at position −9 (Holstege et al., 1997). We did not have a potentially reactive base at this position in the 8gW promoter, so we constructed an 8gW variant with thymine residues from position −8 to −10. Tests of complexes stalled at +8 on this promoter showed reactivity at −8T and −9T (data not shown). Thus, we conclude that the 8gW complex has a 16 base transcription bubble spanning positions −9 to +7 (Figure 2A). The transcription bubble on the 8g2D promoter spanned at least the residues from position −5 to +7. To test whether the upstream edge of the bubble can extend further upstream on 8g2D complexes, we also constructed a variant of 8g2D with thymine residues from position −6 to −8. A KMnO4 sensitivity assay with complexes stalled on this promoter showed that the upstream edge of the bubble extended to position −7, although reactivity at the most upstream T residue was weaker than at other positions (data not shown). Thus, complexes stalled at +7 on the 8g2D promoter have a 12–14 base bubble extending from position −7 to +7. These results are consistent with the upstream edge of the transcription bubble having a fixed position relative to the TATA element (Giardina and Lis, 1993). In Figure 2A, this is shown as 20 bases counted from the first A (boldface) in the TATA box. In 8g2I complexes the bubble was at least 17 bases long. We did not construct a variant promoter to test the exact position of the upstream edge, but if the upstream edge is also 20 bases from TATA, the 8g2I bubble would be 18 bases long. In contrast to the relatively uniform sensitivities of the T residues in the 8g2D and 8gW bubbles, the upstream T residues in the 8g2I bubble were distinctly less reactive as compared with the downstream T residues (gray rectangles, Figure 2A). This suggests that the upstream segment of the 8g2I bubble is on the verge of collapse. In contrast to the much larger bubbles in the other three 8g series complexes, complexes on the 8g4I promoter showed strong upstream permanganate reactivity only to −2, with a very weak signal at −5. Complexes stalled at +8 on the 8g4I promoter have therefore passed the bubble collapse transition. Thus, the transcription bubble sizes on the 8g series of promoters support our initial working model. As bubble size expands, stability drops (compare 8g2D and 8gW, Figure 1). As the upstream segment of the bubble begins to close, stability begins to recover (compare 8gW and 8g2I), and once bubble collapse is complete, stability increases further (compare 8g2I and 8g4I). Note that the maximum bubble size we observe, 17–18

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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.

bases, is consistent with the largest bubbles observed in earlier studies (Holstege et al., 1997). Figure 2B shows the dimensions of transcription bubbles in the 9g series of promoters. In these analyses, we did not construct additional promoters to exactly map the upstream edge, so the results are presented as the minimum bubble size. With the 9gW complex we obtained a 16 base bubble spanning positions −8 to +8. In these complexes the upstream thymine residues (−2, −5, −8) are somewhat less sensitive than the downstream residues, as we observed with the 8g2I complexes (Figure 2A). This suggests that the 9gW complexes are on the verge of bubble collapse. The bubbles on the 9g1I and the 9g2I complexes were much smaller. Only the downstream T residues were strongly reactive; among the upstream T residues, only the −2 position showed any reactivity. Thus, the 9g1I and 9g2I complexes have apparently passed the bubble collapse transition, which is consistent with the higher stability of the 9g1I and 9g2I complexes relative to the 9gW complexes. Bubble Collapse Requires Both a Minimum Bubble Size and a Minimum Transcript Length For the 6g series of promoters, we did not expect to see evidence of bubble collapse, since the stabilities of these complexes declined continuously as the TATA to

+1 spacing increased (Figure 1). As shown in Figure 3, the bubble sizes in these complexes were in accordance with this prediction. The minimum bubble sizes in the 6g2D and 6gW complexes were 10 and 13 bases, respectively. As in the case of the 8g series, we created modified versions of 6g2D and 6gW with additional upstream T residues. Use of these promoters allowed us to place the upstream bubble edges for 6g2D at −7 and for 6gW at −9 (data not shown). The minimum bubble sizes for 6g2I and 6g4I are 15 and 17 bases; the bubble in each case is probably one base longer but we did not confirm this directly. The important point is that the bubble is fully open in both of these (comparatively unstable) complexes, in contrast to the results with the 8g2I and 9gW complexes (Figure 2). We believe it is significant that the upstream segment of the bubble shows no evidence of closure in the 6g4I complex. The bubble in this case is 17–18 bases long, which is the same size as the bubble in the 8g2I complex and longer than the 16–17 base bubble in the 9gW complex. However, the bubbles in 8g2I and 9gW are already closing, as noted above. If we assume that the initial (precollapse) upstream edge of the transcription bubble is always set 20 bases from the TATA box, then 6g4I, 8g2I, and 9g1I complexes should all have bubbles of the identical size. However, while 9g1I has clearly passed through the bubble collapse transition (and is

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Figure 4. Dependence of Transcript Elongation on dATP for the 8g Promoters

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.

stable) and 8g2I is in midtransition (and is becoming stable), the bubble in 6g4I remains completely open and the complex is relatively unstable. We conclude that the length of the transcript influences bubble collapse, and in particular, the RNA must reach a minimum length for collapse to occur. Since bubble collapse does not take place with 6g4I complexes that contain either 5 nt (ApC or ATP-initiated) or 6 nt (CpA-initiated) RNAs, but can occur in 8g complexes with 7 nt RNAs, the transcript must be at least 7 bases long to trigger collapse. Completion of Bubble Collapse Appears to Mark the End of the Promoter Clearance Transition The mechanistic basis of the requirement for TFIIH helicase activity during the earliest stages of transcript elongation has not been clearly established. Perhaps the energy stored in the initial bubble is expended, at bubble collapse, in reorganizing the complex into the elongation form, at which point the assistance of a helicase is no longer necessary. We tested the idea that bubble collapse coincides with the loss of the energy requirement for efficient transcript elongation using the 8g series of promoters. Complexes assembled with purified transcriptional components were initiated in G-less reactions with CpA primers, gently rinsed to remove NTPs, and then chased to the A stop at +23 in

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 ␮M each of CTP, UTP, and GTP, ±40 ␮M 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).

the presence or absence of dATP to support helicase activity. The results with 8gW (Figure 4) were those expected from earlier studies (Dvir, 2002). Elongation through the region w10–15 bases downstream of +1 was inefficient. This barrier was partially overcome by the addition of dATP to the chase reactions, which increased the yield of the 23-mer by 1.8-fold. The barrier to elongation was even greater with the 8g2D complexes. A greater proportion of complexes failed during elongation, and an even greater stimulation of 23-mer synthesis (2.2-fold) was obtained with dATP. In both the 8g2D and 8gW complexes, the upstream segment of the transcription bubble is open. In the 8g2I complex, this region is closing (Figure 2); significantly, transcript elongation in the absence of dATP is much more efficient and the stimulation of 23-mer synthesis by dATP is considerably less (1.2-fold) on 8g2I, as compared with 8g2D and 8gW. Remarkably, the 8g4I complex has no energy requirement for transcript elongation. Essentially all of the complexes chase from 8 to 23 and there is no stimulation of 23-mer accumulation by dATP. Thus, at least for 8-mer complexes on the 8g series of promoters, the role of helicase in assisting transcript elongation is reduced or eliminated exactly coincident with the collapse of the upstream segment of the transcription bubble. We repeated the experiment shown in Figure 4 with all of the promoters in the 6g and 9g series, as well as with complexes halted at +10, +12, and +14. The results are summarized in Figure 5, along with the status of the transcription bubbles in the complexes. These results are generally consistent with the trend observed with the 8g complexes: as the transcription bubble extends up to its maximum size, stimulation by dATP is reduced. Stimulation is very low or absent for those complexes in which bubble collapse has taken place.

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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.

should not occur with bubble templates in the absence of TFIIB. It is possible to test this idea, since we had shown earlier (Keene and Luse, 1999) that pol II will initiate transcription on bubble templates in the absence of initiation factors if a dinucleotide primer is employed. When we performed dinuclotide-primed transcription of the −9/−1 bubble template in the absence of TFIIB (or in the absence of TBP, which should prevent loading of TFIIB), we found that while transcription complex assembly was inefficient, those polymerases that initiated did not pause at all in the +7 to +9 region (Figure 6A, lanes 11–14). If displacement of the B finger of TFIIB is critical for pol II to transcribe beyond +7, then alterations in the B finger would be expected to affect this stage of promoter clearance. To test this we employed a variant of human TFIIB in which residue Arg66 in the B finger domain is changed to leucine. This R66L variant is analogous to the yeast R78L mutant, whose phenotype includes alterations in the start site of transcription for several yeast genes (Faitar et al., 2001). The effect of the amino acid change in R66L is thought to involve elimination of a stabilizing salt bridge with a glutamate residue on the opposite side of the B finger (E62 in yeast or E51 in the human protein; see Pinto et al., 1994; Bushnell et al., 2004). In control reactions with wild-type TFIIB, the ability of pol II to transcribe the bubble template to the G stop at +20 was completely dependent on the addition of dATP. Strong pausing was observed at +7 to +9 (Figure 6B, lanes 3 and 4) even in the presence of dATP. In contrast, with the R66L variant (lanes 7 and 8), some 21-mer was made in the absence of dATP, and a much smaller proportion of polymerases in the reaction without dATP paused at +7 to +9 (31%, versus 70% with wild-type TFIIB). Discussion

A Role for TFIIB in Promoter Clearance How is the energy stored in the growing transcription bubble used during bubble collapse? One way to approach this question is to study transcription with templates in which the two DNA strands are mispaired over the region of the initial transcription bubble. Such bubble templates allow efficient transcript initiation in the absence of TFIIE and TFIIH (Tantin and Carey, 1994; Pan and Greenblatt, 1994), but they cannot undergo bubble collapse. When we synthesized RNA with pol II and purified transcription factors from a template mispaired from −9 to −1, we found that promoter clearance, though strongly stimulated by dATP, remained inefficient even in the presence of the energy source (Figure 6A, lanes 3 and 4). In particular, strong pauses were observed at +7 through +9. These pauses were completely absent when a conventional double-stranded template with the identical template strand sequence was transcribed in the presence of dATP (Figure 6A, lanes 1 and 2). The locations of the +7 to +9 pauses are interesting since they occur just beyond the point at which the advancing 5# end of the RNA is expected to initially encounter the B finger of TFIIB (Bushnell et al., 2004). This suggested to us that bubble collapse could be driving the removal of the B finger from the RNA exit channel. This idea predicts that the +7 to +9 pauses

The transition from initiation to elongation is one of the least understood aspects of RNA polymerase II function. We show here that bubble collapse, the abrupt reannealing of the upstream segment of the initial transcription bubble, appears to mark the end of the clearance process. We demonstrate that bubble collapse depends on two properties of the transcription complex: growth of the transcription bubble to about 18 bases and extension of the transcript to at least 7 nt in length. We also show that TFIIB has an important effect on promoter clearance. By establishing the linkage among transcript length, transcription bubble size, and helicase dependence, our results have unified many earlier observations in these areas (Dvir et al., 1997; Holstege et al., 1997; Yan and Gralla, 1997; Kumar et al., 1998). The demonstration of the central role of TFIIB in promoter clearance significantly extends previous results, in that it provides one specific substrate for the energydependent reorganization of the transcription complex during the clearance transition. The central importance of bubble collapse in the progression from initiation to elongation is illustrated by the properties of the 8g series of complexes. Each of the four 8g complexes we studied contains the identical RNA-DNA hybrid. When transcription is initiated

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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 ␮M UTP, and 1 ␮M [α-32P]CTP with or without 20 ␮M dATP for 5 min at 30°C, followed by an additional 2 min incubation with 20 ␮M 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 wildtype TFIIB (lanes 1–4) or the R66L variant of TFIIB (lanes 5–8). Transcription was performed as in (A).

with CpA, this hybrid is 8 bp long, essentially the length of the hybrid in the transcript elongation complex (Westover et al., 2004). However, the properties of the four 8g complexes are very different (Figures 1 and 4). The parameter that predicts the stability and helicase dependence for a given complex is the position of that complex on the pathway to bubble collapse (Figure 5). Complexes that are well upstream of collapse (8g2D) are stable but very dependent on helicase assistance during elongation. Complexes that are near the transition are unstable (8gW), while complexes in the process of closure (8g2I) have recovered some stability and lost much of their helicase dependence. Complexes that have completely passed the collapse transition (8g4I) are stable, but, in contrast to precollapse complexes, they are also fully independent of helicase during elongation. What Is the Molecular Mechanism of Promoter Clearance? There are many interactions in the PIC that could ultimately impede transcript elongation. As noted above, a segment of TFIIB extends into the polymerase through the presumed RNA exit channel, placing the B finger domain adjacent to the active site and in the path of the advancing 5# end of the RNA (Bushnell et al., 2004; Chen and Hahn, 2004). The XPB helicase subunit of TFIIH interacts with DNA just downstream of the RNA polymerase (Douziech et al., 2000; Kim et al., 2000; Spangler et al., 2001). It seems reasonable to assume that this downstream contact must ultimately be disrupted to allow RNA polymerase to advance. The subunits of TFIIF make extensive contacts over the surface of pol II in the PIC (Chung et al., 2003), and recent studies indicate that TFIIF interacts with TFIIB as well (Chen and Hahn, 2004). In short, an extensive reorganization of the transcript initiation complex should be required to achieve promoter clearance. Since energy is consumed during this same stage of transcription outside of RNA synthesis (Yan and Gralla, 1999), it is appropriate to refer to this reorganization as remodeling (Forget et al., 2004). We propose that bubble collapse marks the end of

this remodeling process, allowing the polymerase to functionally clear the promoter. This is shown schematically in Figure 7. As the polymerase advances, energy is stored within the complex and then expended, at clearance, to transform the complex into the transcript elongation form. A key feature is that the upstream edge of the initial transcription bubble remains stationary, rather than advancing along with the point of bond formation. Thus, as the bubble extends, the energy used to melt the initial bubble is retained within the complex. During the preclearance phase of transcription, additional energy is required as the polymerase begins to disrupt initiation-specific interactions. This energy is partially provided by the helicase activity of TFIIH, consistent with the ATP dependence of elongation at this stage (reviewed in Dvir, 2002) and the observation (Yan and Gralla, 1999) that ATP is required to maintain the transcription bubble in complexes stalled before promoter clearance. Just prior to bubble collapse, stress within the complex would presumably reach a maximum level, consistent with the reduced stability of these complexes. Once a critical threshold is reached, the upstream segment of the bubble reanneals and the stored energy is expended in reorganizing the complex, which is once again stable. How is the growing transcription bubble accommodated within the transcription complex during the clearance process? One could imagine that pol II retains its upstream contacts while stretching downstream as RNA is synthesized (“inchworming”). Alternatively, if the polymerase’s dimensions remain fixed, the template strand must be looped out, or scrunched, as transcription proceeds (see Brieba and Sousa [2001] and references therein). The diagram in Figure 7 suggests a constant pol II footprint prior to clearance, but in fact we have no evidence from our work to favor one model over the other. This will be an important subject for future investigation. If clearance depended only on the extension of the bubble to 17–18 bases, then bubble collapse and clearance should have at least begun in the 6g4I complex. However, in this complex the bubble is fully open over its entire length (Figure 4). The transcript in these com-

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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.

plexes (5 or 6 nt) is probably too short to allow interaction of the B finger of TFIIB with the RNA (Bushnell et al., 2004). This suggests that the initial displacement of the B finger by the advancing RNA is essential to begin bubble collapse. However, complexes on templates where bubble collapse cannot occur are strongly impeded in transcript elongation from +7 to +9 unless TFIIB is absent from the reaction (Figure 6). The extent of pausing from +7 to +9 can be altered by a single amino acid change in the B finger of TFIIB (Figure 6B). Thus, an important part of the remodeling associated with bubble collapse may involve displacement of TFIIB from the RNA exit channel. There is a clear parallel between the displacement of bacterial sigma factor from the RNA exit channel by the nascent RNA (Murakami et al., 2002; Vassylyev et al., 2002) and the fate of TFIIB during the initial stages of transcript elongation. In particular, we note the similarity between the absence of pausing at +7 to +9 in pol II reactions lacking TFIIB (Figure 6A, lanes 11 and 12) and the result obtained (Murakami et al., 2002) with E. coli RNA polymerase and a sigma factor which lacks the domain (region 3.2) expected to occupy the RNA exit channel in the bacterial polymerase. Murakami and colleagues found that transcription with the truncated sigma factor essentially eliminates the tendency of the bacterial RNA polymerase to abortively initiate RNAs of 10 nt or less (Murakami et al., 2002). The results presented here indicate that slight alterations in the spacing of the TATA box and the transcription start site can have profound effects on the stability and helicase dependence of transcription complexes during the initial phase of RNA synthesis by pol II. The TATA-to-start spacing is 30 bp in the wild-type Ad ML promoter, which contains consensus TATA and initiator motifs (Smale and Kadonaga, 2003). A nonexhaustive survey of promoters containing recognizable TATA and initiator elements reveals a range of spacings from 26 (α-fetoprotein) to 34 (histone 2A) bp. It will be interesting to determine whether clearance on promoters with the smallest or largest TATA-initiator spacing obeys the same rules that we observed with Ad ML. A greater challenge will be to extend these analyses to promoters that do not contain TATA elements. Is the upstream edge of the transcription bubble fixed in these promoters as well? Our model would predict that this is true, in order to store energy to drive promoter clearance,

but entirely different mechanisms may apply for nonTATA promoters. Finally, it is tempting to speculate that variations in promoter architecture which lead to differences in the clearance process may also lead to differential responses to transcriptional activators and repressors. Experimental Procedures Reagents and Transcription Templates Ultrapure NTPs were purchased from Amersham Biosciences, ApC dinucleotide from Sigma, 32P-labeled NTPs (800 Ci/mmol) from PerkinElmer Life Sciences, and streptavidin-coated magnetic beads from Promega. CpA was obtained as a custom synthesis from Dharmacon. All promoters were based on the pML20-40 Ad ML promoter (Pal and Luse, 2003). Transcription templates were made by PCR using the same set of primers, with the upstream primer biotinylated, as described (Pal et al., 2001). In Vitro Transcription Using HeLa Nuclear Extracts Preinitiation complexes were assembled using HeLa nuclear extract and bead-attached templates in a final volume of 25 ␮l as described (Pal et al., 2001). Initial G-less reactions were done at 30°C for 1 min with 1 mM ApC or CpA or 75 ␮M ATP, plus 10 ␮M UTP, 20 ␮M dATP, and 1 ␮M [α-32P]CTP. The bead-attached complexes were rinsed twice with ice-cold M5 buffer (20 mM Tris [pH 7.9], 70 mM KCl, 10 mM MgCl2, 1 mM DTT, 0.2 mM EDTA, 5% glycerol) and resuspended in 20 ␮l of M5. Chase reactions were carried out at 30°C for 2 min with 200 ␮M each of CTP, UTP, GTP, and 40 ␮M dATP. RNAs were purified and analyzed as described (Pal et al., 2001). Transcription with Purified Factors Recombinant (FLAG-tagged) general transcription factors (GTFs) TFIIB, TFIIE, and TBP were expressed and purified as described (Chiang and Roeder, 1995; Wu and Chiang, 1998). Recombinant (hexahistidine-tagged) wild-type and R66L variant TFIIB (used in Figure 6) were expressed and purified essentially as described (Bangur et al., 1999) and stored in 20 mM HEPES-KOH (pH 7.6), 20% glycerol, 150 mM KCl, 1 mM DTT, 1 mM EDTA, 0.1 mM ZnCl2, 0.1 mM PMSF. Recombinant human TFIIF subunits were expressed, purified, and assembled as described (Wang et al., 1993). The TFIIH used for these studies was made from HeLa nuclear extract as described (Maldonado et al., 1996) with the following modifications: The DE52 fraction (eluting at 0.35 M KCl) was loaded on to a 1 ml mono Q column at 0.1 M KCl. The column was developed with a linear gradient from 0.1 to 0.8 M KCl in 20 mM Tris (pH 7.9), 1 mM DTT, 0.2 mM EDTA, 0.5 mM PMSF, and 20% glycerol. The TFIIH activity eluted between 250 and 350 mM KCl. RNA polymerase II from calf thymus was purified as described (Keene and Luse, 1999). The reactions in Figure 6 were done with human pol II, which was extracted as described (Maldonado et al., 1996) from the HeLa

Promoter Clearance by RNA Polymerase II 109

nuclear pellets and then purified by chromatography on DE52, heparin Sepharose, and Mono Q. PICs were assembled in 20 ␮l at 30°C for 40 min by incubating 2.5 ng bead-attached template with roughly 2.5 ng TFIIB, 10 ng TFIIF, 10 ng TFIIE, 3 ng TBP, 0.75 ␮l TFIIH (Mono Q fraction), and 15 ng RNA polymerase II in 20 mM Tris (pH 7.9), 70 mM KCl, 10 mM MgCl2, 0.2 mM DTT, 0.2 mM EDTA, 10% glycerol, and 0.5 mg/ ml BSA. After assembly, PICs were kept on ice until used. Transcription was initiated with 0.5 mM ApC (unless noted otherwise in the figure) plus 10 ␮M UTP, 20 ␮M dATP, and 1 ␮M [α-32P]CTP. RNA products were purified and analyzed as described above. KMnO4 Sensitivity Assay PICs were assembled using purified pol II and GTFs as above except that end-labeled templates (w2000 cpm/ng) were used, at a concentration 1/4 of that given above. Two minutes after the addition of NTPs, 2 ␮l of 5 mM KMnO4 was added; 30 s later, 2 ␮l of 2.5 M β-mercaptoethanol was added followed by 25 ␮l of 40 mM EDTA, 0.5 mg/ml proteinase K, and 1% SDS. Samples were incubated for 10 min at 65°C and purified by phenol/chloroform extraction and ethanol precipitation. DNAs were cleaved at modified T residues by resuspension in 100 ␮l of 1 M piperidine, 1 mM EDTA, 1 mM EGTA, and 25 ␮g/ml herring sperm DNA, followed by incubation at 90°C for 30 min. DNA was recovered by ethanol precipitation and resolved on 7% polyacrylamide gels (19:1 acrylamide:bisacrylamide) containing 7 M urea. Acknowledgments We thank Cheng-Ming Chiang and Shwu-Yuan Wu for providing reagents and guidance on transcription factor purification and use, Danny Reinberg and Subhrangshu Mandal for a gift of TFIIH and advice on TFIIH and pol II purification, and Suzie Luse for pol II preparation. We also thank Andrea Újvári and Louise Steele for valuable comments during the course of these studies. This work was supported by grants GM 29487 (to D.S.L.) and GM 51124 (to A.S.P.) from the National Institutes of Health. Received: December 12, 2004 Revised: April 20, 2005 Accepted: May 19, 2005 Published: June 30, 2005 References Armache, K.-J., Kettenberger, H., and Cramer, P. (2003). Architecture of initiation-competent 12-subunit RNA polymerase II. Proc. Natl. Acad. Sci. USA 100, 6964–6968. Asturias, F.J. (2004). Another piece in the transcription initiation puzzle. Nat. Struct. Mol. Biol. 11, 1031–1033. Bangur, C.S., Faitar, S.L., Folster, J.P., and Ponticelli, A.S. (1999). An interaction between the N-terminal region and the core domain of yeast TFIIB promotes the formation of TATA-binding proteinTFIIB-DNA complexes. J. Biol. Chem. 274, 23203–23209. Brieba, L.G., and Sousa, R. (2001). T7 promoter release mediated by DNA scrunching. EMBO J. 20, 6826–6835. Buratowski, S. (2000). Snapshots of RNA polymerase II transcription initiation. Curr. Opin. Cell Biol. 12, 320–325.

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