Pcf11 Is a Termination Factor in Drosophila that Dismantles the Elongation Complex by Bridging the CTD of RNA Polymerase II to the Nascent Transcript

Molecular Cell 21, 65–74, January 6, 2006 ª2006 Elsevier Inc.

DOI 10.1016/j.molcel.2005.11.002

Pcf11 Is a Termination Factor in Drosophila that Dismantles the Elongation Complex by Bridging the CTD of RNA Polymerase II to the Nascent Transcript Zhiqiang Zhang1 and David S. Gilmour1,* 1 Department of Biochemistry and Molecular Biology Center for Gene Regulation The Pennsylvania State University University Park, Pennsylvania 16802

Summary The mechanism by which Pol II terminates transcription in metazoans is not understood. We show that Pcf11 is directly involved in termination in Drosophila. dPcf11 is concentrated at the 30 end of the hsp70 gene in cells, and depletion of dPcf11 with RNAi causes Pol II to readthrough the normal region of termination. dPcf11 also localizes to most transcribed loci on polytene chromosomes. Biochemical analysis reveals that dPcf11 dismantles elongation complexes by a CTDdependent but nucleotide-independent mechanism and that dPcf11 forms a bridge between the CTD and RNA. This bridge appears to be crucial because an antiCTD antibody, which also dismantles the elongation complex, is found to bridge the CTD to RNA. dPcf11 was observed to inhibit transcription at low, but not high, nucleotide levels, suggesting that dPcf11 dismantles paused elongation complexes. These results provide a biochemical basis for the dependency of termination on pausing and the CTD in metazoans. Introduction Termination of Pol II transcription is an essential step in gene expression, but the mechanism is poorly understood. Besides its requirement for recycling use of Pol II, the choice of termination site can influence the availability of splice sites and polyadenylation sites in premRNA. It has been estimated that half of the mRNAs in humans utilize alternate polyadenylation sites (Iseli et al., 2002), and this can affect the location, stability, and coding potential of the transcripts. Pol II molecules that fail to terminate can inhibit function of downstream promoters by displacing proteins from the DNA (Greger et al., 1998; Greger and Proudfoot, 1998). This so-called transcription interference can serve to regulate expression of some genes (Martens et al., 2004). Pol II termination is coupled to polyadenylation by the polyadenylation signal in the nascent transcript. Two models have been proposed to explain this coupling (Buratowski, 2005; Luo and Bentley, 2004). According to the torpedo model, cleavage of the nascent transcript, which precedes polyadenylation, generates an uncapped end on the residual transcript engaged with Pol II. This uncapped end is an entry point for a 50 to 30 exonuclease that chases down the Pol II and induces termination. The torpedo model received recent support with the finding that mutation of a 50 to 30 exonuclease, called Rat1, causes Pol II to readthrough terminators in yeast (Kim et al., 2004b). Depletion of the homologous *Correspondence: [email protected]

protein Xrn2 from human cells also impairs termination on a transiently transfected b globin gene (West et al., 2004). An alternative model, originally called the antiterminator model but now generalized as the allosteric model (Luo and Bentley, 2004), posits that the polyadenylation signal in the nascent transcript causes an allosteric change in Pol II that decreases the processivity of the elongation complex (EC). This could be due to the dissociation of an antiterminator from the EC or association of a factor that depresses processivity. Until recently, the strongest support for the allosteric model was provided by circumstances in which termination occurs in the absence of the cleavage reaction (Osheim et al., 1999, 2002; Sadowski et al., 2003). Under these circumstances, the torpedo model for termination cannot apply, as there is no entry point for the 50 to 30 exonuclease. Recently, we discovered that a yeast protein called Pcf11 dismantles a yeast Pol II EC (Zhang et al., 2005). This reaction depends on the CTD of Pol II, thus providing a possible reason for why deletion of the CTD impairs termination in human cells (McCracken et al., 1997). The CTD corresponds to the unusual C-terminal domain of the largest Pol II subunit and is composed of multiple copies of a heptapeptide with the consensus YSPTSPS (Buratowski, 2003; Meinhart et al., 2005). yPcf11 appears to dismantle the EC by bridging the CTD to the nascent transcript (Zhang et al., 2005). In yeast, mutations in yPcf11 impair both termination and polyadenylation (Sadowski et al., 2003). yPcf11 is in a complex called CF1A, which is involved in processing the 30 end of mRNAs (Gross and Moore, 2001a). CF1A recognizes part of the tripartite polyadenylation signal in the GAL7 gene (Gross and Moore, 2001b), thus providing a possible basis for how the polyadenylation signal might recruit or regulate the activity of yPcf11. Human Pcf11 is in a complex with at least 15 other polypeptides, and the complex is required for 30 end processing in vitro (de Vries et al., 2000). The hPcf11 complex interacts with CF1m and CPSF, two proteins that recognize the polyadenylation signal in the nascent transcript (de Vries et al., 2000; Venkataraman et al., 2005). Nothing is known about the role of hPcf11 in termination. Given the results that termination can occur prior to nascent transcript cleavage (Osheim et al., 1999, 2002), and our discovery that yPcf11 could be the engine that drives the termination reaction in yeast (Zhang et al., 2005), we wanted to determine if Pcf11 was involved in termination in a metazoan. Results dPcf11 Associates with Transcriptionally Active Loci on Polytene Chromosomes To obtain insight into the function of dPcf11 in Drosophila, we used immunofluorescence microscopy to analyze the distribution on polytene chromosomes of a protein that had 37% amino acid sequence identity with the CTD interacting domain of yPcf11 (Meinhart and Cramer, 2004). Antibodies were raised against an N-terminal

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Figure 1. Immunofluorescence Analysis of dPcf11 on Polytene Chromosomes (A and B) Polytene chromosomes from nonheat-shocked larvae stained with antibody against Pol II and dPcf11, respectively. Loci were mapped based on the DNA staining pattern (Figure S1A). Pol II was detected with the ARNA-3 antibody, which binds the largest subunit outside the CTD (Kramer et al., 1980). (C and D) Polytene chromosomes from heatshocked larvae stained with antibody against Pol II and dPcf11. (E) Western blot analysis of whole-cell lysates from salivary glands and S2 cells with antibody raised against dPcf11. The band at w62 kDa is close to the predicted mobility of Pcf11. (F) High magnification view of the 87A locus in a stretched polytene chromosome from a heat-shocked larva. The schematic shows the two divergently transcribed copies of hsp70 that generate the heat-shock puff at 87A. The bottom two panels show individual staining for Pol II and dPcf11. Color images of (A)–(D) and (F) are provided in Figure S1.

polypeptide encompassing amino acids 1–238. In accordance with one form of dPcf11 predicted from cDNA sequence information, Western blotting detected a single polypeptide of w60 kDa in whole-cell lysates from Drosophila salivary glands and tissue culture cells (Figure 1E). Immunofluorescence analysis revealed that dPcf11 was concentrated at a small number of loci on polytene chromosomes. For chromosomes from either nonheatshocked larvae (Figures 1A and 1B, and Figure S1A available in the Supplemental Data with this article online) or heat-shocked larvae (Figures 1C and 1D and Figure S1B), all locations staining intensely for dPcf11 also stained intensely for Pol II. The chromosomes from heatshocked larvae provide the strongest indication that dPcf11 is involved in transcription. Heat shock causes the induction of a small collection of genes whose locations on the chromosomes are known (Ashburner and Bonner, 1979). dPcf11 is concentrated at every one of these locations (Figure 1D and Figure S1B). In addition to the naturally occurring genes, dPcf11 was also detected on a heat shock-inducible transgene located at 87E (Wu et al., 2003). Interestingly, dPcf11 was not detected at the myriad of locations on the chromosome from nonheat-shocked larvae that modestly stain with antibody against Pol II (Figures 1A and 1B and Figure S1A). Many of these locations are likely to contain Pol II molecules that have paused as a result of the negative elongation factor NELF (Wu et al., 2003, 2005). Locus 87A on some polytene chromosomes from heat-shocked larvae exhibited very intriguing patterns

of dPcf11 and Pol II staining. 87A contains two copies of the hsp70 gene that are divergently transcribed (Figure 1F and Figure S1C). When this region was stretched during preparation of the chromosomes, the staining pattern for each protein appeared as two bands. Antibody against Pol II stained two regions toward the center of the locus, whereas antibody against dPcf11 stained two regions toward the periphery. These patterns of staining suggest that dPcf11 is concentrated toward the 30 ends of each copy of hsp70. ChIP Analysis Reveals that dPcf11 Is Concentrated at the 30 End of the hsp70 Gene and that Depletion of dPcf11 with RNAi Causes Transcriptional Readthrough To determine if dPcf11 was involved in transcription termination in Drosophila cells, we used a combination of RNA interference (RNAi) and chromatin immunoprecipitation (ChIP). ChIP revealed that dPcf11 was concentrated at the 30 end of hsp70. Figure 2B shows data from one ChIP experiment, and Figure 2C quantifies the results from two independent experiments. The primers used for the analyses in Figure 2 detected the three copies of hsp70 located at 87C. All five copies of hsp70 are nearly identical and induced by heat shock (Gilmour et al., 1986). The presence of dPcf11 at the 30 end of hsp70 was revealed by the marked enrichment of the +2433 to +2601 region over a control region in the dPcf11 immunoprecipitate (Figure 2B, cf. lanes 4 and 20; Figure 2C). In addition, lower levels of dPcf11 were detected near the 50 end of the gene and in a region

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Figure 2. Analysis of dPcf11 Function In Vivo Using ChIP and RNAi (A) Western blot analysis for dPcf11 and a control protein, CBP80, in LacZ RNA-treated (lane 1), dPcf11 RNAi-treated (lane 2), and untreated (lane 3) Drosophila cells. CBP80 is the largest subunit of the cap binding protein (Izaurralde et al., 1994) and serves as a loading control. (B) One data set for a ChIP analysis of dPcf11 and Pol II on the hsp70 gene in heat-shocked cells. The bands correspond to radiolabeled DNA PCR amplified from immunoprecipitates of dPcf11 (lanes 5–12 and 17–20) and Pol II (lanes 25–32 and 37–40) from crosslinked cells previously treated with lacZ RNAi (lanes 5–8 and 25–28), dPcf11 RNAi (lanes 9–12 and 29–32), or no RNAi (lanes 17–20 and 37–40). Samples in lanes 13–16 and 33–36 provide a measure of background and represent material immunoprecipitated from untreated cells with dPcf11 preimmune antiserum. Samples in lanes 1–4 and 21–24 represent 2% of the material subjected to immunoprecipitation (input). The band marked with an asterisk is from a region of the genome lacking any known transcription units. The bands delimited by the two vertical bars are PCR products from hsp70 spanning +10 to +181, +1201 to +1364, +2003 to +2166, and +2433 to +2601: each region corresponds respectively to the first through the fourth lane in each group of four lanes. (C and D) Quantification of ChIP data from two independent experiments for hsp70. The polyadenylation signal for hsp70 is at +2326. The error bars indicate the range of values for the two experiments.

preceding the polyadenylation site (Figure 2B, lanes 17 and 19; Figure 2C) but appeared to be absent from the region in the middle of the gene (Figure 2B, lane 18; Figure 2C). Several controls indicate that the signals detected were due to crosslinking of dPcf11. The signals were reduced when the immunoprecipitations were done with preimmune serum (Figure 2B, lanes 13–16; Figure 2C). Also, treatment of cells with RNAi targeting dPcf11 mRNA reduced the level of dPcf11 detected on the hsp70 gene (Figure 2B, lanes 9–12; Figure 2C). Western blot analysis indicated that the dPcf11 RNAi greatly diminished the level of dPcf11 in the cell (Figure 2A, lane 2). The distribution of Pol II on hsp70 was monitored by ChIP using antibody against the Rpb3 subunit (Adelman et al., 2005). Although depletion of dPcf11 had no significant impact on the density of Pol II across the body of hsp70, it increased the level of Pol II crosslinking downstream from the polyadenylation signal by w3-fold (Figure 2B, cf. lanes 28, 32, and 40; Figure 2D). This increase in Pol II downstream from the polyadenylation signal indicates that the efficiency of termination was impaired by depletion of dPcf11. This conclusion is further supported by RT-PCR, which detects RNA from

the +2433 to +2601 region in cells depleted of dPcf11, but not in control cells (Figure S2). dPcf11 Dismantles a Pol II EC by a CTD-Dependent but ATP-Independent Mechanism We assembled an EC with purified Drosophila Pol II and tested the effect of adding various derivatives of dPcf11 produced in E. coli. Pol II was allowed to initiate transcription on a tailed, G less cassette template (Zhang et al., 2004). In the presence of the UpG dinucleotide, ATP, CTP, and radioactive UTP, Pol II initiates transcription at the tailed end of the template and pauses at the end of the G less cassette (Figure 3A). These ECs remain active, as they will resume transcription upon addition of GTP (Zhang et al., 2004). Also, the nascent transcript is sensitive to RNase A, but not RNase H, indicating that the nascent transcript has not hybridized to the template strand of DNA along its entire length, as sometimes occurs with other tailed templates (Zhang et al., 2004). When the ECs were analyzed on a native gel, two bands were detected (Figure 3B, lane 1). The upper band contains Pol IIA with an intact CTD; the lower band contains Pol IIB that has lost its CTD during the purification of the Pol II (Zhang et al., 2004). The Pol IIB EC provides

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an internal control for monitoring CTD-dependent disruption of the EC. Treatment of the ECs with full-length dPcf11 (1–574) selectively eliminated the Pol IIA EC (Figure 3B, lane 5). As we previously reported (Zhang et al., 2004), the same thing occurred when the ECs were treated with a CTD antibody (Figure 3B, lane 6). By testing various derivatives of dPcf11, dismantling activity was mapped to the N terminus encompassed by amino acids 1–149. This region is homologous to the region of yPcf11 recently shown to dismantle a yeast EC (Zhang et al., 2005). The disappearance of the Pol IIA complex was not simply the consequence of binding protein to the CTD. CstF50 is a subunit of the polyadenylation factor CstF and had previously been shown to associate with the CTD (McCracken et al., 1997). In contrast to dPcf11, treatment of the ECs with dCstF50 shifted the mobility of the Pol IIA EC (Figure 3C). To further analyze the activity of dPcf11, we examined the effects of dPcf11 on a mixture of Pol IIA and IIB ECs that were assembled on immobilized DNA templates. dPcf11 derivatives encompassing amino acids 1–149, 1–283, and 1–574 (full length) caused the immobilized complexes to release half of the transcript, whereas buffer or a derivative of Pcf11 lacking the N-terminal region did not release transcript (Figure 3D, lanes 1–10). No transcripts were released when Pol IIB ECs were challenged with dPcf11 (Figure 3D, lanes 11–16), so the portion of transcripts released from the Pol IIA/IIB mixture must be derived from Pol IIA ECs. In accord with this, Western blotting revealed that dPcf11 1–283 dissociated Pol IIA, but not Pol IIB, from the immobilized template (Figure 3E, lanes 7 and 8). In agreement with the gel shift data, dCstF50 failed to dissociate either form of Pol II from the template (Figure 3E, lanes 5 and 6). Taken together, the results demonstrate that the N-terminal region of dPcf11 completely dismantles the Pol IIA EC by a mechanism that requires the CTD but no nucleotides.

Figure 3. dPcf11 Dismantles Pol IIA, but Not Pol IIB ECs (A) Depiction of the EC used in these studies. (B) Native gel analysis of Pol II ECs (IIA EC and IIB EC). ECs were formed and then treated with buffer (lane 1), various derivatives of dPcf11 (lanes 2–5), or the CTD antibody (lane 6). Finally, the integrity of the ECs were evaluated by native gel electrophoresis. The numbers associated with each derivative of dPcf11 identify the amino acid region constituting the protein. dPcf11 1–574 is the full-length protein. (C) Native gel analysis of Pol II ECs treated with dCstF50 (lane 2) or dPcf11 1–283 (lane 3). (D) Transcripts released from immobilized Pol II ECs after incubation with derivatives of dPcf11. A mixture of Pol IIA and Pol IIB ECs (lanes 1–10) or Pol IIB ECs (lanes 11–16) alone were formed on immobilized DNA templates. Immobilized ECs were incubated for 15 min with various derivatives of dPcf11 and then separated into bound (‘‘B’’) and released (‘‘R’’) fractions. Radiolabeled transcripts were isolated and analyzed on a denaturing gel. (E) Measurement of Pol II released from immobilized Pol IIA/IIB ECs.

dPcf11 Can Form a Bridge between the CTD and RNA Recently, we discovered that yPcf11 can form a bridge between the CTD and RNA and that this bridge appears to be important for the dismantling reaction (Zhang et al., 2005). To determine if dPcf11 dismantles the EC by a similar mechanism, we first tested various derivatives of dPcf11 for CTD binding activity. All the derivatives that were found to dismantle the EC were also found to associate with the CTD (Figure 4A, lanes 2, 4, and 5). Next, we tested dPcf11 1–283 for RNA binding activity (this derivative was used because its yield from E. coli was significantly higher than the others). UV crosslinking was used to detect protein-RNA interactions. Pcf11, in the presence or absence of GST-CTD, was incubated with radiolabeled RNA and then UV irradiated to induce crosslinks between RNA and associated proteins. The

Immobilized Pol IIA/IIB ECs were incubated with buffer, dCstF50, or dPcf11 1–238 for 15 min and then separated into B and R fractions. The largest subunit of Pol II was detected in each fraction by Western blotting with the ARNA-3 antibody, which binds outside the CTD (Kramer et al., 1980). IIa is the largest subunit derived from Pol IIA, and IIb is the largest subunit derived from Pol IIB. Lanes 1 and 2 show Pol IIB and the mixture of Pol IIA and Pol IIB. Pol IIB was prepared as previously described (Zhang et al., 2004).

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RNA was degraded with RNase, and proteins crosslinked to the residual RNA tags were identified after SDS-PAGE. dPcf11, but not GST-CTD, crosslinked to RNA (Figure 4C, lanes 1–3). mPcf11, a mutant form of dPcf11 found to completely inhibit the dismantling activity of dPcf11 (data not shown), also failed to crosslink to RNA (Figure 4, lanes 4 and 5). This mutation has amino acids 75, 76, and 77 mutated to alanines and corresponds to a mutation in yPcf11 that impairs termination (Sadowski et al., 2003; Zhang et al., 2005). Because dPcf11 associated with both the CTD and RNA, we tested if dPcf11 could bridge the CTD to RNA. GST-CTD was immobilized and then incubated with radiolabeled RNA in the presence or absence of dPcf11. In the absence of dPcf11 or the presence of mPcf11 1– 238, no RNA associated with the CTD (Figure 4D, lanes 3 and 6). In the presence of dPcf11, w20% of the input RNA was observed to bind to the immobilized CTD (Figure 4D, lane 5). These results indicate that dPcf11 can bridge the CTD to RNA. A CTD Antibody with Dismantling Activity Has the Capacity to Bridge the CTD to RNA As shown in Figure 3B (lane 6) and previously reported (Zhang et al., 2004), a monoclonal antibody that bound specifically to the CTD of Pol II dismantled the EC. If the formation of a bridge between the CTD and RNA was essential to the dismantling reaction, we predicted that the CTD antibody should associate with RNA. UV crosslinking showed that this was indeed the case. RNA crosslinked specifically to the light chain of the antibody with an efficiency similar to that of dPcf11 (Figure 4E, lanes 1–3). The absence of crosslinking to the heavy chain indicates that the method detects a specific association with the light chain rather than simply the random collision of molecules. We also found that the CTD antibody bridges RNA to the immobilized form of the CTD (Figure 4D, lane 8). Because dPcf11 and the CTD antibody can both bridge the CTD to RNA, yet their structures are unrelated, we conclude that a crucial step in the dismantling reaction is formation of a bridge between the CTD and RNA. This conclusion is further supported by our observation that another monoclonal antibody that binds RNA (data not shown), but associates with the body of Pol II, fails to dismantle the EC (Zhang et al., 2004). Figure 4. dPcf11 and a CTD Antibody Can Bridge the CTD to RNA (A) GST-CTD pull-down assays to detect interaction between dPcf11 and the CTD. GST (lane 1) or GST-CTD (lanes 2–5) were immobilized on glutathione Sepharose and then incubated with various derivatives of dPcf11. Bound protein was eluted and analyzed by Western blotting with antibody against the histidine tag on dPcf11. (B) Coomassie blue-stained gel of the dPcf11 preparations used in (A). (C) UV crosslinking assay to detect protein-RNA interactions. One microgram of each of the proteins listed at the top of each lane was incubated with radiolabeled RNA, followed by UV irradiation. RNA was degraded with RNase, and radioactively tagged proteins were detected after SDS-PAGE. mPcf11 1–238 has a three amino acid mutation that inactivates its dismantling activity (data not shown). The weak signal in lane 3 that comigrates with GST-CTD was not reproducible. (D) Analysis of CTD-RNA bridging activity. Glutathione Sepharose was first incubated with buffer (lane 1), GST (lanes 2 and 9), GSTCTD (lanes 3, 5, 6, and 8), dPcf11 1–238 (lane 4), or CTD antibody (lane 7) for 30 min. After several washes, the beads were incubated

dPcf11 Binds Preferentially to a Small Region of the CTD Unlike the CTDs of yeast and human Pol II in which 19 of 26 and 21 of 52 of the respective heptad repeats exactly match the consensus YSPTSPS, only two of the 42 with buffer (lanes 1–4, and lane 7), dPcf11 1–238 (lane 5), mPcf11 1– 238 (lane 6), or CTD antibody (lane 8 and 9). Beads were again washed and then incubated with radiolabeled RNA. After this last incubation, beads were extensively washed to remove unbound RNA. Radiolabeled RNA was recovered from the beads and analyzed on a denaturing polyacrylamide gel. Lane 10 (Input) shows 50% of the total RNA added to each sample. (E) UV crosslinking analysis to detect CTD antibody-RNA interactions. Two dPcf11 derivatives or the CTD antibody were incubated with radiolabeled RNA and subjected to crosslinking analysis. Lanes 1–3 show the radioactively tagged proteins. Lanes 4–6 show Coomassie blue staining of proteins in the gel.

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tom panel of Figure 5B shows that approximately equivalent quantities of each the GST-CTD derivatives were loaded onto the glutathione Sepharose for these binding assays. dPcf11 Acts on Paused ECs Previously, we had observed that the CTD antibody inhibited transcription at low, but not high, nucleotide concentrations (Zhang et al., 2004). Low nucleotide concentrations promote pausing by the EC. As pausing appears to precede termination in vivo (Orozco et al., 2002; Park et al., 2004), we tested if there were any differences in the way dPcf11 affected elongation reactions at two nucleotide concentrations. The results in Figure 6 show that dPcf11 inhibited elongation by Pol IIA at low, but not high, nucleotide concentrations. Fifty percent inhibition of the Pol IIA/IIB mixture occurred when transcription was done at a low nucleotide concentration in the presence of dPcf11 1–238 (Figure 6A, cf. lanes 1 and 2; Figure 6B, column 1a), whereas no inhibition occurred at a high nucleotide concentration (Figure 6A, cf. lanes 5 and 6; Figure 6B, column 2a). Neither mPcf11 nor dPcf11 151–574 affected transcription at either nucleotide concentration (Figure 6B, columns 1b, 1c, 2b, and 2c). Also, no inhibition of transcription was observed at either nucleotide concentration when transcription was performed with purified Pol IIB (Figure 6B, columns 3a and 4a). Together, these results indicate that dPcf11 inhibits transcription elongation by Pol IIA under conditions that promote pausing. Discussion Figure 5. dPcf11 Associates Preferentially with a Part of the CTD Containing the Only Two Heptad Motifs that Match the Consensus (A) The amino acid sequence of the CTD of Drosophila Pol II. The sequence is displayed to emphasize the heptads matching the consensus YSPTSPS. Asterisks demarcate the only two heptads that precisely match the consensus heptad. Each of the four copies of the sequence PSYSPTSP shown in bold matches the part of the CTD that contacts yPcf11 in a crystal (Meinhart and Cramer, 2004). The vertical lines demarcate regions of the CTD that have been fused to GST. (B) GST-CTD pull-down assays to detect interaction between dPcf11 and subregions of the CTD. Derivatives of the CTD fused to GST (lanes 1–4) or GST alone (lane 5) were immobilized on glutathione Sepharose. CTD1 spans the entire CTD, whereas CTD2, CTD3, and CTD4 are demarcated in (A). Each immobilized GST derivative was incubated with dPcf11 1–238. Bound Pcf11 was eluted and analyzed by Western blotting with antibody against the histidine tag on dPcf11 1–238 (top). The bottom panel shows the Coomassiestained GST derivatives that had been immobilized and subsequently run on SDS-PAGE.

We have provided evidence that dPcf11 is directly involved in Pol II termination in Drosophila. Immunofluorescence microscopy and ChIP indicated that dPcf11 was concentrated at the 30 end of the hsp70 gene, and depletion of dPcf11 from Drosophila cells increased the level of Pol II normally detected downstream from the polyadenylation signal of hsp70. In addition, we found that the N-terminal region of dPcf11 completely dismantled an elongation complex. This last result sets dPcf11 apart from all other proteins that have been implicated in Pol II termination and is strong evidence that dPcf11 is directly involved in termination. The detection of dPcf11 at most highly transcribed loci in polytene chromosomes suggests that dPcf11 is involved in termination at many genes. dPcf11 provides a basis for connecting three key aspects of termination: the CTD, the polyadenylation signal, and pausing.

repeats in the Drosophila CTD exactly match the consensus (Figure 5A). Hence, we were interested in determining where dPcf11 associated with the CTD, as this might provide insight into the dismantling mechanism. Pull-down assays with immobilized derivatives of the Drosophila CTD fused to GST revealed that dPcf11 preferentially associated with the region encompassing the two consensus heptads. dPcf11 was observed to bind equally well to an intact version of the CTD and to a truncated version of the CTD containing the two conserved heptads but bound markedly less well to parts of the CTD that flanked the conserved heptads (Figure 5B, top, compare lanes 1 and 3 with lanes 2 and 4). The bot-

CTD Dependence A crucial step in the termination reaction mediated by dPcf11 appears to be the formation of a bridge between the CTD and the nascent transcript, as this is the only thing common to the CTD antibody and dPcf11, both of which dismantled the EC. Additional support for the importance of the bridge comes from our analysis of yeast Pcf11: mutations impairing RNA binding or CTD binding each inhibited the dismantling reaction (Zhang et al., 2005). In addition, the dismantling reaction can be inhibited by hybridizing a DNA oligonucleotide to the nascent transcript in the region just outside from where RNA exits Pol II (Zhang et al., 2005). Presumably,

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Figure 6. dPcf11 Inhibits Transcription by Pol IIA at Low, but Not High, Nucleotide Concentrations Pol II was incubated with the tailed template in the presence or absence of dPcf11 derivatives for 5 min prior to the addition of ATP, CTP, and radioactive UTP. Transcription was allowed to proceed for 20 min, and the resulting transcripts were analyzed on denaturing gels. (A) Transcripts produced by the Pol IIA/IIB mixture or Pol IIB at high and low nucleotide concentrations. High nucleotide conditions: 500 mM ATP, 500 mM CTP, and 25 mM a-32P UTP. Low nucleotide conditions: 25 mM ATP, 25 mM CTP, and 10 mM a-32P UTP. (B) Quantification of transcript levels. The graph shows the amount of transcript formed in the presence of each dPcf11 derivative divided by the amount of transcript produced in the absence of dPcf11. Error bars represent standard deviations from the mean for three independent experiments.

the oligonucleotide blocks formation of the bridge by interfering with the Pcf11-RNA interaction. Because the CTD antibody and Pcf11 are structurally unrelated, it is unlikely that the dismantling reaction involves Pcf11 directly recognizing part of the body of Pol II. How the formation of the bridge disrupts the elongation complex is a mystery. One possibility is that constraining the CTD or the RNA causes either of these or Pcf11 itself to contact the RNA exit channel in a way that destabilizes the EC. RNA-protein interactions in the RNA exit channel of bacterial RNA polymerase contribute to pausing and termination (Toulokhonov and

Landick, 2003). The molecular contacts at the RNA exit channel of the Pol II EC may be uniquely suited for allosteric control of the EC, because it was observed that Rho, which normally functions in termination in bacteria, disrupts Pol II ECs, but not Pol I or Pol III ECs (Lang et al., 1998). Rho moves along RNA in a 50 to 30 direction, so it probably collides with the region of Pol II at the RNA exit channel. dPcf11 seems to interact with a relatively small region of the Drosophila CTD. This is in contrast with the yeast and human CTDs where Pcf11 could in principal coat almost all of the yeast CTD and half of the human CTD. The results from Drosophila suggest that the bridge does not have to form close to the body of the Pol II molecule to dismantle the EC. The binding of dPcf11 to the Drosophila CTD may not be dictated by the heptad per se but by a slightly larger motif that appears four times in the region where dPcf11 bound the CTD (Figure 5A, bold letters). This motif, PSYSPTSP, corresponds to the region of a peptide composed of two consensus heptads that was contacted by yPcf11 in a crystallized complex (Meinhart and Cramer, 2004). Phosphorylation of the CTD could influence the activity of Pcf11. Phosphorylation of serine 2 in the CTD appears to increase the affinity of yPcf11 for the CTD (Barilla et al., 2001; Licatalosi et al., 2002; Sadowski et al., 2003). Importantly though, yPcf11 binds the unphosphorylated CTD (Barilla et al., 2001; Licatalosi et al., 2002; Sadowski et al., 2003), and there is evidence in yeast indicating that the CTD of Pol II is dephosphorylated just prior to termination (Buratowski, 2005). ChIP data indicate that the level of serine 2 phosphorylation increases as Pol II moves from the 50 to the 30 end of the hsp70 gene (Boehm et al., 2003), and the same occurs on several yeast genes (Ahn et al., 2004). This rising level of serine 2 phosphorylation could contribute to the recruitment of Pcf11 near the 30 end of the gene. However, the phosphates on the CTD might also antagonize the ability of Pcf11 to form a bridge with the nascent transcript due to electrostatic repulsion. The CTD phosphatase Ssu72 has been implicated in termination (Krishnamurthy et al., 2004; Steinmetz and Brow, 2003). Ssu72 might participate in termination by removing phosphates from the CTD so the bridge can form between the CTD and RNA. Polyadenylation Signal Dependence Our results show that dPcf11 is concentrated near the polyadenylation signal of hsp70, similar to what was observed for several genes in yeast (Kim et al., 2004a). Though Pcf11 binds RNA, it seems unlikely that Pcf11 alone recognizes the polyadenylation signal in the nascent transcript. First, we observed equivalent crosslinking to two unrelated RNAs, neither of which contained a polyadenylation signal (data not shown). Second, amino acids 1–149 of Pcf11 lack any known RNA recognition motifs. Nevertheless, Pcf11 appears to have a surface that interacts specifically with RNA, because mutating one amino acid in yeast Pcf11 impaired RNA binding without affecting CTD binding (Zhang et al., 2005). Yeast Pcf11 is part of a complex called CF1A, which contains three other subunits (Gross and Moore, 2001a). One subunit, Rna15, recognizes part of the polyadenylation signal, thus providing a way to recruit yPcf11 to the end of the gene after the polyadenylation signal has been

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transcribed (Gross and Moore, 2001b). Human Pcf11 is part of a complex called CFIIA, which itself does not appear to recognize the polyadenylation signal (de Vries et al., 2000). CFIIA, however, interacts with CPSF and CFIm, two proteins that recognize different parts of the polyadenylation signal in humans and that are involved in pre-mRNA 30 end processing (Venkataraman et al., 2005; Zhao et al., 1999). If CPSF and CFIm are involved in recruiting Pcf11 to the 30 end of genes in metazoans, regulation is needed to prevent Pcf11 from prematurely terminating transcription. ChIP detects both CPSF and CFIm well upstream of the polyadenylation site in the human G6PD gene (Venkataraman et al., 2005), and earlier studies indicated that CPSF could be recruited to the 50 end of genes through association with TFIID (Dantonel et al., 1997). Dependency on Pause Sites The location of pause sites will be a key parameter in dictating where Pcf11 dismantles the elongation complex. As long as the EC is moving, it resists the action of Pcf11. We suspect this resistance arises because the RNA reeling out of an actively moving EC interferes with physical interactions that might be required for the dismantling reaction. There are ample data to indicate that pause sites are involved in selection of termination sites (see Plant et al. [2005] and references therein). Diverse mechanisms could be used by the cell to cause the EC to pause. These include the presence of pause sites that are intrinsic to the DNA sequence (Palangat et al., 2004). Intrinsic pauses are found scattered throughout almost any stretch of DNA, so this could account for the stochastic selection of termination sites downstream from a polyadenylation signal. Specific proteins bound to the DNA could cause pausing as appears to be the case for the MAZ protein (Ashfield et al., 1994). Finally, nucleosomes cause ECs to pause (Izban and Luse, 1991). This could explain why chromatin remodeling factors appear to act as terminators (Alen et al., 2002). Conclusion The dependence of the Pcf11 dismantling reaction on pausing and the CTD provide possible explanations for why these two things are important for termination. The specificity of termination probably arises from the combinatorial actions of factors that control pausing, the association of Pcf11 with the CTD, and the association of Pcf11 with the nascent transcript. Our results provide direct support for an allosteric model of termination but certainly do not preclude possible contributions from an RNA exonuclease after cleavage of the nascent transcript. One possibility that we previously proposed is that the exonuclease shortens the residual nascent transcript, forcing Pcf11 to bind close to the RNA exit channel (Zhang et al., 2005). Experimental Procedures Expression and Purification of dPcf11 and dCstF50 cDNA clones RE43027 and LD24780 encoding, respectively, dPcf11 and dCstF50 were obtained from the Drosophila Genomics Resource Center. Sequences encoding various parts of dPcf11 or all of dCstF50 were cloned into the pET-28-(a) expression vector (Novagen). Plasmids were transformed into E. coli BL21(DE3), and 6-histidine tagged versions of each protein were expressed and

then purified on Ni-NTA-agarose according to the manufacturer’s instructions (Qiagen). Immunofluorescence Analysis of Polytene Chromosomes dPcf11 antibody was generated in a rabbit and a guinea pig by immunizing with dPcf11 1–283. Affinity purification of the antibody and immunofluorescence microscopy were done as previously described (Wu et al., 2003). Similar results were obtained with both antibodies. Pol II was detected with ARNA-3, a monoclonal antibody that associates with the largest Pol II subunit in a region outside the CTD (Kramer et al., 1980). Depletion of dPcf11 from Drosophila S2 Cells with RNAi Depletion of dPcf11 with RNAi was done similarly to Clemens et al. (2000). dPcf11 template was produced by PCR from a dPcf11 cDNA clone with the primers 50 -GAATTAATACGACTCACTATAGGGAGAG CGGTCCGGTAAGATCAC-30 and 50 -GAATTAATACGACTCACTATA GGGAGATCAACCACATACGGTCCC-30 , and a lacZ template was produced with the primers 50 -GAATTAATACGACTCACTATAGGGA GATGAAAGCTGGCTACAGGA-30 and 50 -GAATTAATACGACTCACT ATAGGGAGAGCAGGCTTCTGCTTCAAT-30 . Double-stranded RNA was synthesized from the DNA templates by using the MEGAscript T7 kit (Ambion). To treat Drosophila S2 cells with RNAi, cells were grown to a density of 1 3 106 cells/ml at 24ºC in Schneider’s Drosophila medium (GIBCO-BRL) supplemented with 10% heat-inactivated fetal bovine serum. Cells were diluted in half with the same medium, and 25 ml of cells were put in a 75 cm2 tissue culture flask. The cells were incubated for 2 hr, and then the medium was replaced with fresh medium containing 60 ug/ml of double-stranded RNA but no fetal bovine serum. Cells were grown for 3 days and then subject to experimental analyses. ChIP Analysis Twenty-five milliliters of S2 cells (2 3 106 cells/ml) was heat shocked for 20 min at 37ºC. After the heat shock, 675 ml of 37% formaldehyde stock solution was added directly to the culture medium to give a final concentration of 1%. Subsequent manipulations were done as described by Wu et al. (2003) to yield 3 ml of soluble chromatin. Three-hundred microliters of soluble chromatin was used for each immunoprecipitation along with 3 ml of Rpb3 antiserum (Adelman et al., 2005), 5 ml of anti-dPcf11 serum, or 5 ml of preimmune serum. The resulting DNA was dissolved in 30 ml of TE (10 mM Tris-Cl [pH 8], 1 mM EDTA). To prepare DNA representing input DNA, 30 ml of soluble chromatin was combined with 470 ml of elution buffer. Reversal of formaldehyde crosslinks and isolation of the DNA was done as for DNA eluted from the immunoprecipitates. The final DNA was dissolved in 30 ml of TE and further diluted with TE as needed. PCR reactions for detecting specific DNA in each reaction were done similarly to those previously found to be in the quantitative range of the reaction (Wu et al., 2003). There are multiple copies of the hsp70 genes located at 87A and 87C, and previous studies show that they are comparably expressed in vivo (Gilmour et al., 1986). The following primers were used to amplify different regions of the hsp70 genes located at 87C: +10 to +181, 50 -AAACAAGCAAAGTGAACACG TC-30 and 50 -TCAATAATTACTTCTTGGCAGATTTC-30 ; +1201 to +1364, 50 -ACGATGCCAAGATGGACAA-30 and 50 -CTGCACAGCAGC TCCGTAT-30 ; +2003 to + 2166, 50 -GGAGTTCGACCACAAGATGG-30 and 50 -AGTCGACCTCGACTGTG-30 ; and +2433 to +2601, 50 -GCGAT GGAGAGTTGGCGCCG-30 and 50 -GGGCGAACGGCGGTTCC-30 . An internal standard was included in all the PCR reactions. The standard corresponded to the region from 8366490 to 8366713 in the Drosophila genome and was amplified with the primers 50 -TTG CTGTCTGGCGTAAATTG-30 and 50 -TCAGCGGAGTCCTTGAAGAT30 . Ten microliters of each PCR reaction was run on a 6% nondenaturing acrylamide gel. Gels were fixed in 10% acetic acid for 20 min and then dried. Radioactivity was detected in the dried gel by a phosphoimager (Molecular Dynamics). The intensity of each band was quantified by volume analysis using ImageQuant software. Formation of Pol II ECs Methods describing the purification of Drosophila Pol II, formation of ECs, their analysis by native gel electrophoresis, and their analysis on immobilized templates have been described (Zhang et al., 2004).

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Expression and Purification of GST-CTD Fusion Proteins GST-CTD derivatives were generated by subcloning sequences encoding all or part of the Drosophila CTD derived from clone LD43558 (Drosophila Genomics Resource Center) into the plasmid pGEX 4T-3 (Amersham Biosciences). E. coli BL21 cells containing expression plasmids were grown in LB containing 100 mg/ml Ampicillin at 37ºC to an OD600 of 0.5–0.8, and expression was induced by the addition of IPTG to 1.0 mM. After 3 hr at 37ºC, cells were harvested and lysed, and proteins were purified by affinity chromatography on glutathione Sepharose (Amersham Biosciences) according to the manufacturer’s instructions. GST Pull-Down Assays for dPcf11 Binding Two-hundred nanograms of GST or GST-CTD derivatives was bound to 15 ml of glutathione Sepharose (Pharmacia) in 150 mM KCl-HGEDPX (HGEDPX corresponds to 25 mM HEPES [pH 7.6], 10% glycerol, 0.1 mM EDTA, 1 mM DTT, 0.1 mM PMSF, 1 mM sodium bisulfite, and 1 mM benzamidine HCl) for 1 hr at 4ºC. Beads were washed five times with 500 mM KCl-HGEDPX and suspended in 100 ml 150 mM KCl-HGEDPX. One-hundred nanograms of dPcf11 was incubated with each aliquot of beads for 1 hr at 4ºC. Beads were washed four times with 500 ml 150 mM KCl-HGEDPX for 5 min at 4ºC. Proteins were eluted in SDS buffer and analyzed by SDS-PAGE. dPcf11 was detected by Western blotting with antibody against the histidine tag, and GST derivatives were detected by Coomassie staining. Analysis of RNA-Protein Interactions Detection of RNA-protein interactions by UV crosslinking was done as previously described (Zhang et al., 2005). One microgram of each protein and 300,000 cpm of radiolableled RNA was used in each reaction. Detection of CTD-RNA Bridging Activity To test if dPcf11 or the CTD antibody (8WG16, [Thompson et al., 1989]) could bridge RNA to the CTD, 10 mg of each GST derivative was incubated with 50 ml glutathione Sepharose at room temperature for 30 min in 150 mM KCl, 50 mM HEPES (pH 7.6), 1.0 mM MnCl2, 12% glycerol, and 0.5 mM DTT. Beads were extensively washed and then incubated with 3 mg of protein (dPcf11 or CTD antibody) for 15 min. Again, beads were extensively washed. Finally, each sample was incubated with 300,000 cpm of RNA at room temperature for 20 min followed by extensive washing to remove unbound material. To elute RNA, the beads were incubated at 42ºC for 30 min in 100 ml of 20 mM EDTA (pH 8), 200 mM NaCl, 1% SDS, 250 mg/ml yeast RNA, and 0.1 mg/ml proteinase K. RNA was phenol extracted, ethanol precipitated, and analyzed on a denaturing polyacrylamide gel. Transcription Reactions at High and Low Nucleotide Concentrations Transcription reactions were performed in 15 ml containing 200 mM KCl, 50 mM HEPES (pH 7.6), 1.0 mM MnCl2, 12% glycerol, 0.5 mM DTT, 0.5 mM UpG, 20 U RNasin, 1.0 ml of purified Pol II (1 microgram), and 100 ng of tailed G less template (Zhang et al., 2004). Pol II was preincubated with the template in the presence or absence of dPcf11 for 5 min to allow Pol II to bind the end of the DNA. For transcription under high nucleotide conditions, a cocktail of nucleotides was added to yield final nucleotide concentrations of 500 mM ATP, 500 mM CTP, 25 mM UTP, and 1 mCi a-32P UTP/reaction. For transcription under low nucleotide conditions, the final concentrations were 25 mM ATP, 25 mM CTP, 10 mM UTP, and 1 mCi a-32P UTP/reaction. Each reaction mixture was incubated at 21ºC for 25 min. RNA was isolated and analyzed on a denaturing polyacrylamide gel. Supplemental Data Supplemental Data include two figures and can be found with this article online at http://www.molecule.org/cgi/content/full/21/1/65/ DC1/. Acknowledgments We thank Maria Horvat-Gordon for doing preliminary immunofluorescence analyses. We also thank John T. Lis (Cornell University)

for providing Rpb3 antiserum. This work was supported by research grant GM47477 from the National Institutes of Health to D.S.G. Received: August 10, 2005 Revised: October 17, 2005 Accepted: November 2, 2005 Published: January 5, 2006 References Adelman, K., Marr, M.T., Werner, J., Saunders, A., Ni, Z., Andrulis, E.D., and Lis, J.T. (2005). Efficient release from promoter-proximal stall sites requires transcript cleavage factor TFIIS. Mol. Cell 17, 103–112. Ahn, S.H., Kim, M., and Buratowski, S. (2004). Phosphorylation of serine 2 within the RNA polymerase II C-terminal domain couples transcription and 30 end processing. Mol. Cell 13, 67–76. Alen, C., Kent, N.A., Jones, H.S., O’Sullivan, J., Aranda, A., and Proudfoot, N.J. (2002). A role for chromatin remodeling in transcriptional termination by RNA polymerase II. Mol. Cell 10, 1441–1452. Ashburner, M., and Bonner, J.J. (1979). The induction of gene activity in Drosophilia by heat shock. Cell 17, 241–254. Ashfield, R., Patel, A.J., Bossone, S.A., Brown, H., Campbell, R.D., Marcu, K.B., and Proudfoot, N.J. (1994). MAZ-dependent termination between closely spaced human complement genes. EMBO J. 13, 5656–5667. Barilla, D., Lee, B.A., and Proudfoot, N.J. (2001). Cleavage/polyadenylation factor IA associates with the carboxyl-terminal domain of RNA polymerase II in Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. USA 98, 445–450. Boehm, A.K., Saunders, A., Werner, J., and Lis, J.T. (2003). Transcription factor and polymerase recruitment, modification, and movement on dhsp70 in vivo in the minutes following heat shock. Mol. Cell. Biol. 23, 7628–7637. Buratowski, S. (2003). The CTD code. Nat. Struct. Biol. 10, 679–680. Buratowski, S. (2005). Connections between mRNA 30 end processing and transcription termination. Curr. Opin. Cell Biol. 17, 257–261. Clemens, J.C., Worby, C.A., Simonson-Leff, N., Muda, M., Maehama, T., Hemmings, B.A., and Dixon, J.E. (2000). Use of doublestranded RNA interference in Drosophila cell lines to dissect signal transduction pathways. Proc. Natl. Acad. Sci. USA 97, 6499–6503. Dantonel, J.C., Murthy, K.G., Manley, J.L., and Tora, L. (1997). Transcription factor TFIID recruits factor CPSF for formation of 30 end of mRNA. Nature 389, 399–402. de Vries, H., Ruegsegger, U., Hubner, W., Friedlein, A., Langen, H., and Keller, W. (2000). Human pre-mRNA cleavage factor II(m) contains homologs of yeast proteins and bridges two other cleavage factors. EMBO J. 19, 5895–5904. Gilmour, D.S., Pflugfelder, G., Wang, J.C., and Lis, J.T. (1986). Topoisomerase I interacts with transcribed regions in Drosophila cells. Cell 44, 401–407. Greger, I.H., and Proudfoot, N.J. (1998). Poly(A) signals control both transcriptional termination and initiation between the tandem GAL10 and GAL7 genes of Saccharomyces cerevisiae. EMBO J. 17, 4771– 4779. Greger, I.H., Demarchi, F., Giacca, M., and Proudfoot, N.J. (1998). Transcriptional interference perturbs the binding of Sp1 to the HIV-1 promoter. Nucleic Acids Res. 26, 1294–1301. Gross, S., and Moore, C. (2001a). Five subunits are required for reconstitution of the cleavage and polyadenylation activities of Saccharomyces cerevisiae cleavage factor I. Proc. Natl. Acad. Sci. USA 98, 6080–6085. Gross, S., and Moore, C.L. (2001b). Rna15 interaction with the A-rich yeast polyadenylation signal is an essential step in mRNA 30 -end formation. Mol. Cell. Biol. 21, 8045–8055. Iseli, C., Stevenson, B.J., de Souza, S.J., Samaia, H.B., Camargo, A.A., Buetow, K.H., Strausberg, R.L., Simpson, A.J., Bucher, P., and Jongeneel, C.V. (2002). Long-range heterogeneity at the 30 ends of human mRNAs. Genome Res. 12, 1068–1074.

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Izaurralde, E., Lewis, J., McGuigan, C., Jankowska, M., Darzynkiewicz, E., and Mattaj, I.W. (1994). A nuclear cap binding protein complex involved in pre-mRNA splicing. Cell 78, 657–668. Izban, M.G., and Luse, D.S. (1991). Transcription on nucleosomal templates by RNA polymerase II in vitro: inhibition of elongation with enhancement of sequence-specific pausing. Genes Dev. 5, 683–696. Kim, M., Ahn, S.H., Krogan, N.J., Greenblatt, J.F., and Buratowski, S. (2004a). Transitions in RNA polymerase II elongation complexes at the 30 ends of genes. EMBO J. 23, 354–364. Kim, M., Krogan, N.J., Vasiljeva, L., Rando, O.J., Nedea, E., Greenblatt, J.F., and Buratowski, S. (2004b). The yeast Rat1 exonuclease promotes transcription termination by RNA polymerase II. Nature 432, 517–522. Kramer, A., Haars, R., Kabisch, R., Will, H., Bautz, F.A., and Bautz, E.K. (1980). Monoclonal antibody directed against RNA polymerase II of Drosophila melanogaster. Mol. Gen. Genet. 180, 193–199. Krishnamurthy, S., He, X., Reyes-Reyes, M., Moore, C., and Hampsey, M. (2004). Ssu72 is an RNA polymerase II CTD phosphatase. Mol. Cell 14, 387–394. Lang, W.H., Platt, T., and Reeder, R.H. (1998). Escherichia coli rho factor induces release of yeast RNA polymerase II but not polymerase I or III. Proc. Natl. Acad. Sci. USA 95, 4900–4905. Licatalosi, D.D., Geiger, G., Minet, M., Schroeder, S., Cilli, K., McNeil, J.B., and Bentley, D.L. (2002). Functional interaction of yeast premRNA 30 end processing factors with RNA polymerase II. Mol. Cell 9, 1101–1111. Luo, W., and Bentley, D. (2004). A ribonucleolytic rat torpedoes RNA polymerase II. Cell 119, 911–914. Martens, J.A., Laprade, L., and Winston, F. (2004). Intergenic transcription is required to repress the Saccharomyces cerevisiae SER3 gene. Nature 429, 571–574. McCracken, S., Fong, N., Yankulov, K., Ballantyne, S., Pan, G., Greenblatt, J., Patterson, S.D., Wickens, M., and Bentley, D.L. (1997). The C-terminal domain of RNA polymerase II couples mRNA processing to transcription. Nature 385, 357–361. Meinhart, A., and Cramer, P. (2004). Recognition of RNA polymerase II carboxy-terminal domain by 30 -RNA-processing factors. Nature 430, 223–226. Meinhart, A., Kamenski, T., Hoeppner, S., Baumli, S., and Cramer, P. (2005). A structural perspective of CTD function. Genes Dev. 19, 1401–1415. Orozco, I.J., Kim, S.J., and Martinson, H.G. (2002). The poly(A) signal, without the assistance of any downstream element, directs RNA polymerase II to pause in vivo and then to release stochastically from the template. J. Biol. Chem. 277, 42899–42911. Osheim, Y.N., Proudfoot, N.J., and Beyer, A.L. (1999). EM visualization of transcription by RNA polymerase II: downstream termination requires a poly(A) signal but not transcript cleavage. Mol. Cell 3, 379– 387. Osheim, Y.N., Sikes, M.L., and Beyer, A.L. (2002). EM visualization of Pol II genes in Drosophila: most genes terminate without prior 30 end cleavage of nascent transcripts. Chromosoma 111, 1–12. Palangat, M., Hittinger, C.T., and Landick, R. (2004). Downstream DNA selectively affects a paused conformation of human RNA polymerase II. J. Mol. Biol. 341, 429–442. Park, N.J., Tsao, D.C., and Martinson, H.G. (2004). The two steps of poly(A)-dependent termination, pausing and release, can be uncoupled by truncation of the RNA polymerase II carboxyl-terminal repeat domain. Mol. Cell. Biol. 24, 4092–4103. Plant, K.E., Dye, M.J., Lafaille, C., and Proudfoot, N.J. (2005). Strong polyadenylation and weak pausing combine to cause efficient termination of transcription in the human Ggamma-globin gene. Mol. Cell. Biol. 25, 3276–3285. Sadowski, M., Dichtl, B., Hubner, W., and Keller, W. (2003). Independent functions of yeast Pcf11p in pre-mRNA 30 end processing and in transcription termination. EMBO J. 22, 2167–2177. Steinmetz, E.J., and Brow, D.A. (2003). Ssu72 protein mediates both poly(A)-coupled and poly(A)-independent termination of RNA polymerase II transcription. Mol. Cell. Biol. 23, 6339–6349.

Thompson, N.E., Steinberg, T.H., Aronson, D.B., and Burgess, R.R. (1989). Inhibition of in vivo and in vitro transcription by monoclonal antibodies prepared against wheat germ RNA polymerase II that react with the heptapeptide repeat of eukaryotic RNA polymerase II. J. Biol. Chem. 264, 11511–11520. Toulokhonov, I., and Landick, R. (2003). The flap domain is required for pause RNA hairpin inhibition of catalysis by RNA polymerase and can modulate intrinsic termination. Mol. Cell 12, 1125–1136. Venkataraman, K., Brown, K.M., and Gilmartin, G.M. (2005). Analysis of a noncanonical poly(A) site reveals a tripartite mechanism for vertebrate poly(A) site recognition. Genes Dev. 19, 1315–1327. West, S., Gromak, N., and Proudfoot, N.J. (2004). Human 50 / 30 exonuclease Xrn2 promotes transcription termination at co-transcriptional cleavage sites. Nature 432, 522–525. Wu, C.H., Yamaguchi, Y., Benjamin, L.R., Horvat-Gordon, M., Washinsky, J., Enerly, E., Larsson, J., Lambertsson, A., Handa, H., and Gilmour, D. (2003). NELF and DSIF cause promoter proximal pausing on the hsp70 promoter in Drosophila. Genes Dev. 17, 1402–1414. Wu, C.H., Lee, C., Fan, R., Smith, M.J., Yamaguchi, Y., Handa, H., and Gilmour, D.S. (2005). Molecular characterization of Drosophila NELF. Nucleic Acids Res. 33, 1269–1279. Zhang, Z., Wu, C.H., and Gilmour, D.S. (2004). Analysis of Pol II elongation complexes by native gel electrophoresis: evidence for a novel CTD-mediated termination mechanism. J. Biol. Chem. 279, 23223– 23228. Zhang, Z., Fu, J., and Gilmour, D.S. (2005). CTD-dependent dismantling of the RNA polymerase II elongation complex by the pre-mRNA 30 -end processing factor, Pcf11. Genes Dev. 19, 1572–1580. Zhao, J., Hyman, L., and Moore, C. (1999). Formation of mRNA 30 ends in eukaryotes: mechanism, regulation, and interrelationships with other steps in mRNA synthesis. Microbiol. Mol. Biol. Rev. 63, 405–445.