Leading Edge
Perspective
Cell
A Movie of RNA Polymerase II Transcription
Alan CM. Cheung1 and Patrick Cramer1'*
1Gene Center and Department of Biochemistry, Center for Integrated Protein Science Munich (CIPSM), Ludwig-Maximilians-Universität München, Feodor-Lynen-Str. 25, 81377 Munich, Germany 'Correspondence: cramer@lmb.uni-muenchen.de DOI 10.1016/j.cell.2012.06.006
We provide here a molecular movie that captures key aspects of RNA polymerase II initiation and elongation. To create the movie, we combined structural snapshots of the initiation-elongation transition and of elongation, including nucleotide addition, translocation, pausing, proofreading, backtracking, arrest, reactivation, and inhibition. The movie reveals open questions about the mechanism of transcription and provides a useful teaching tool.
RNA polymerase (Pol) II is a 12 subunit enzyme that depends on additional factors for transcription initiation, elongation, and termination (Orphanidesetal., 1996; Reinesetal., 1996; Roeder, 1996; Sims et al., 2004; Svejstrup, 2004; Thomas and Chiang, 2006; Vannini and Cramer, 2012). Transcription initiation begins with the formation of a closed promoter complex, which contains the 10 subunit Pol II core, the Pol II subcomplex Rpb4/7, and the transcription factors (TF) IID (which includes the TATA-box binding protein TBP and TBP-associated factors), TFIIB, TFIIE, TFIIF, and TFIIH.
Isomerisation of the closed to the open promoter complex involves separation of the DNA strands (i.e., DNA "melting") to form an unwound DNA region (the transcription "bubble") and positioning of the emerging template single strand in the active center of Pol II, which allows RNA synthesis to initiate from the transcription start site (Saunders et al., 2006; Wade and Struhl,
2008) . The initially transcribing complex (ITC) is unstable and releases short RNAs during abortive initiation (Luse and Jacob, 1987). When the RNA reaches a critical length, initiation factors are released, and a stable elongation complex (EC) is formed (Hieb et al., 2006; Holstege et al., 1997), which contains a DNA-RNA hybrid of eight to nine base pairs (Kireeva et al., 2000; Gnattetal.,2001).
During transcription elongation, the EC repeatedly performs the nucleotide addition cycle (NAC) to attach a nucleotide to the growing messenger RNA (mRNA) chain by catalyzing DNA template-directed formation of an RNA phosphodiester bond (Brueckner et al., 2009). The EC can also adopt "offline" states when it transiently pauses at certain DNA sequences (Landick, 2006), backtracks and arrests (Cheung and Cramer, 2011; Nudler et al., 1997; Wang et al., 2009), encounters a DNA lesion in the template strand (Brueckner et al., 2007; Damsma et al., 2007; Damsma and Cramer, 2009; Tornaletti, 2009), or misincor-porates a noncomplementary nucleotide (Sydow and Cramer,
2009) . Elongation factors, such as TFIIS and Spt4/5, are required to deal with these obstacles. TFIIS can reactivate an arrested EC by stimulating RNA cleavage (Reines et al., 1989; Rudd et al., 1994), and Spt4/5, which has a bacterial homolog, can increase transcription processivity (i.e., the property of the polymerase to
stay associated with a transcribed template) (Burova et al., 1995; Martinez-Rucobo et al., 2011; Werner, 2012). To learn more about the prokaryotic RNA polymerase, see the Perspective by Nudler (2012) in this issue of Cell.
From Static Structures to a Movie
Crystal structures of multisubunit RNA polymerases from all three domains of life—bacteria (Vassylyev et al., 2002; Zhang et al., 1999), archaea (Hirata et al., 2008; Korkhin et al., 2009), and eukaryotes (Cramer et al., 2000, 2001) —have been determined. Based on these data, the initiation and elongation phases of transcription were delineated over the last decade (Cramer et al., 2008; Hahn, 2004; Lane and Darst, 2010). Most of the structural information was derived from X-ray analysis, but other techniques were also used, including fluorescence resonance energy transfer (FRET), electron microscopy, and protein cross-linking. Here, we integrated structural information on Pol II complexes with nucleic acids and transcription factors into a movie that visualizes Pol II initiation and elongation (Movie S1 available online). The movie was assembled from a collection of crystal structures and models (Table 1) that were combined into functional polymerase complexes (Figures 1 and 2).
All structures and models were superimposed ontothe structure of the 12 subunit Pol II (PDB code 1WCM) (Armache et al., 2005), which was used as a reference. Modeling, prior to animation, was performed with Coot (Emsley et al., 2010). We used UCSF Chimera to generate all animations, labeling, and morphing interpolations between structures (Pettersen et al., 2004), and then we used FFMPEG (http://ffmpeg.org) to encode the resulting image frames into video. Initiation and elongation factors are colored green and orange, respectively, and flexible polypeptide chains are represented by dotted lines. The orientations of Pol II complexes shown in the movie are restricted to either the front or side views used in earlier publications (Cramer et al., 2001) (Figure 2). See Movie S1 online to download forteaching purposes.
Initiation Complex Formation
The movie starts with the formation of the initiation-competent Pol ll-TFIIF complex (Figures 1A and 2A). Binding of the 10
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Table 1. Structures Used to Generate the Transcription Movie			
Structure	Organism	PDB	References
Rpb4/7	S. cerevisiae	1Y14	(Armache et al., 2005)
10 subunit Pol II	S. cerevisiae	1150	(Cramer et al., 2001)
12 subunit Pol II	S. cerevisiae	1WCM	(Armache et al., 2005)
TFIIF dimerization module	H. sapiens	1F3Ua	(Gaiser et al., 2000)
Pol ll-TFIIF complex model	S. cerevisiae	a,b	(Chen etal., 2010)
TBP-TFIIB-DNA complex	H. sapiens/A. thaliana	1VOLc	(Nikolov et al., 1995)
Pol ll-TFIIB complex	S. cerevisiae	3K1FC	(Kostrewa et al., 2009)
Pol II partial open complex with DNA	S. cerevisiae	4A3I	(Cheung et al., 2011)
Minimal initially transcribing complexes	S. cerevisiae	d	(Cheung et al., 2011)
Archaeal RNA polymerase clamp-Spt4/5 complex	P. furiosis	3QQCe	(Martinez-Rucobo et al., 2011)
Eukaryotic Spt4/5	S. cerevisiae	2EXUe	(Guo etal., 2008)
Pol II EC with exiting RNA, upstream DNA duplex, and nontem plate strand	S. cerevisiae	e,f	(Andrecka et al., 2008, 2009)
Posttranslocated complete EC	S. cerevisiae	1Y1Weg	(Kettenberger et al., 2004)
Bacterial EC substrate complex, preinsertion state	T. thermophilus	2PPBS	(Vassylyev et al., 2007b)
EC substrate complex, insertion state	S. cerevisiae	2E2HS	(Wang etal., 2006)
Pretranslocated EC	S. cerevisiae	1I6HS	(Gnattetal., 2001)
a-Amanitin-inhibited EC	S. cerevisiae	2VUMg	(Brueckner and Cramer, 2008)
Bacterial RNA polymerase EC	T. thermophilus	1IW7S	(Vassylyev et al., 2002)
Paused Pol II with frayed RNA	S. cerevisiae	3HOU	(Sydow et al., 2009)
Arrested Pol II with backtracked RNA	S. cerevisiae	3P02	(Cheung and Cramer, 2011)
Pol II reactivation intermediate with TFIIS	S. cerevisiae	3P03	(Cheung and Cramer, 2011)
aHomology modeling was used to generate a model for S. cerevisiae. bModeled from crosslinking data.
''These structures were combined to create open and closed promoter complex models (Kostrewa et al., 2009). dPDB codes 4A3G, 4A3J, 4A3B, 4A3M, 4A3C, 4A3E, 4A3D, 4A3F, 4A3K, and 4A3L. These structures were used to model the Pol ll-Spt4/5 EC (Martinez-Rucobo et al., 2011).
'Positions of the RNA, upstream DNA duplex, and nontemplate strand were determined by using single-molecule FRET. sUsed to model the nucleotide addition cycle.
subunit Pol II core (Cramer et al., 2001) to the Pol II subcomplex Rpb4/7 (Armache et al., 2005) generates the complete, 12 subunit enzyme (Armache et al., 2003; Bushnell and Kornberg, 2003). Rpb4/7 binding stabilizes a closed conformation of the Pol II clamp domain, which only permits passage of single-stranded DNA to the active site (Armache et al., 2003). The complete Pol 11 is apparently relevant for initiation and elongation because Rpb4/7 is required for initiation in vitro (Edwards et al., 1991) and because the complete Pol II is associated with the genome in vivo (Jasiaket al., 2008). Subsequent binding of TFIIF to Pol II generates the complete Pol ll-TFIIF complex. We positioned the dimerization domain of TFIIF on the lobe domain of Pol II based on crosslinking data (Chen et al., 2010; Eichner etal., 2010).
In the next stage of the movie, the Pol ll-TFIIF complex binds the TBP-TFIIB-DNA complex, resulting in a minimal closed promoter complex, in accordance with the classic model for initiation complex formation (Buratowski et al., 1989) (Figures 1B and 2B). The closed complex model was derived by combining crystal structures of the Pol ll-TFIIB complex (Kostrewa et al., 2009; Liu et al., 2010) and the TBP-TFIIB-DNA complex (Kosa et al., 1997; Littlefield et al., 1999; Nikolov et al., 1995; Tsai and Sigler,
2000). This model reveals the central role of TFIIB as a bridge between the promoter and the polymerase. Docking of the TBP-TFIIB-DNA complex onto the Pol ll-TFIIF complex involves the binding of the TFIIB N-terminal ribbon domain to the Pol II dock domain and the binding of the C-terminal TFIIB core domain to the polymerase wall. The TFIIB reader and linker regions connect the N- and C-terminal domains of TFIIB and extend through the Pol II cleft (Figures 1B, 2B, and 2C). The minimal initiation complex in the movie corresponds to the essential transcription machinery in archaea (Qureshi et al., 1997; Werner and Groh-mann, 2011). It is related to the core initiation complex formed by the two other eukaryotic RNA polymerases, Pol I and Pol III, which useTBPand aTFIIB-likefactor (Vannini and Cramer, 2012).
Initiation-Elongation Transition
The next step in transcription is the formation of the open promoter complex, which involves DNA melting and formation of a transcription bubble. DNA melting commences above the active center cleft, ~20 base pairs downstream of the TATA box (Giardina and Lis, 1993; Kostrewa et al., 2009). DNA melting allows the template single strand to reach the active site and the downstream DNA duplex to bind near the jaws of Pol II (Cheung
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B
Rpb4/7
TFIIB ribbon'
TFIIF
Active site Mg
Initiation-competent complete Pol ll-TFIIF complex
Minimal closed promoter complex
Minimal open promoter complex
Initially transcribing complex (ITC)
Exiling
Backtracked ANA
Pol H-Spt4/5 elongation complex
Arrested complex
V
Pol ll-TFIIS reactivation intermediate
Figure 1. Functional Pol II States Described in the Movie
Schematic representation of seven Pol II functional states described in the movie (A, initiation-competent complete Pol ll-TFIIF complex; B, minimal closed promoter complex; C, minimal open promoter complex; D, initially transcribing complex; E, Pol N-Spt4/5 elongation complex; F, arrested complex; G, Pol ll-TFIIS reactivation intermediate). Pol II is depicted in silver, initiation factors in different shades of green, and elongation factors in different shades of orange. The DNA template strand is in dark blue, the nontemplate strand in light blue, and the RNA in red. The colors are consistent with the movie and Figure 2.
et al., 2011) (Figure 2C). The open complex then initiates RNA synthesis from DNA-templated nucleoside triphosphate (NTP) substrates, forming the ITC (Figures 1D and 2D). We animated this phase by using Pol II structures with short DNA-RNA hybrids of two to seven base pairs in length, both in the absence or presence of an NTP (Cheung et al., 2011). Similar structures were obtained by another group (Liu et al., 2011). Initial RNA synthesis may include a transient "tilting" of the short DNA-RNA hybrid in the absence of a bound NTP.
Initial RNA synthesis further involves "scrunching" of the emerging DNA strands at the upstream edge of the bubble (Figures 1D and 2D). This scrunching occurs because the upstream DNA duplex remains on the polymerase surface, whereas the downstream DNA is pulled into the active center as the DNA-RNA hybrid grows. We modeled scrunched DNA strands in accordance with single-molecule experiments of initiating bacterial RNA polymerase (Kapanidis et al., 2006; Revyakin et al., 2006). During abortive transcription, which is not shown in our movie, Pol II maintains contact with the promoter, allowing it to reinitiate RNA synthesis. Once the RNA-DNA hybrid contains eight base pairs, it is stably associated with Pol II (Kireeva et al., 2000). As the RNA extends farther, contacts to the promoter and initiation factors are broken (Van Dyke et al., 1988; Zawel et al., 1995), resulting in promoter escape and formation of the EC. We animated this transition by fading out TFIIB and TBP and adding RNA in the exit channel upstream of the hybrid (Andrecka et al., 2008; Vassylyev et al., 2007a). We also illustrate the rewinding of upstream DNA (Holstege et al., 1997; Pal et al., 2005) by fading in the upstream DNA duplex that emanates at right angles from the downstream duplex (Andrecka et al., 2009).
Transcription Elongation
In the next stage of the movie, the complete EC (Kettenberger et al., 2004) is bound by the elongation factor Spt4/5 (Klein et al., 2011; Martinez-Rucobo et al., 2011). Spt4/5 binds to the polymerase clamp and is located adjacent to the nontemplate strand of the transcription bubble (Figure 2E). Spt4/5 can associate only upon promoter escape because its binding site is occupied in the initiation complex. The N-terminal NusG homology domain of Spt5 closes the active center cleft, apparently locking in the nucleic acids and enhancing EC processivity. The view then zooms in on the active site to observe the NAC, similar to a previous animation (Brueckner et al., 2009).
During the NAC, an NTP substrate first binds to an open active center conformation, adopting a preinsertion state (Kettenberger et al., 2004; Vassylyev et al., 2007b) that was modeled based on a high-quality bacterial EC structure (Vassylyev et al., 2007b). The NTP then moves slightly to occupy the insertion site as the trigger loop folds to close the active center (Vassylyev et al., 2007b; Wang et al., 2006). Closure of the active site around the NTP generates contacts that are required for correct NTP selection and leads to catalytic nucleotide incorporation and RNA extension (Cheung et al., 2011; Vassylyev et al., 2007b; Wang et al., 2006). Release of pyrophosphate may cause trigger loop unfolding and opening of the active site (Feig and Burton, 2010). Subsequent nucleic acid translocation may follow a two-step mechanism. In the first step, the polymerase trigger loop and bridge helix cooperate to shift the nascent hybrid base pair out of the active site. This leads to translocation of the next template base into a pretemplating position above the bridge helix (Brueckner and Cramer, 2008). In the second step,
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Figure 2. Snapshots from the Movie
Representative still images from the movie corresponding to the seven functional states shown in Figure 1 (A, initiation-competent complete Pol ll-TFIIF complex; B, minimal closed promoter complex; C, minimal open promoter complex; D, initially transcribing complex; E, Pol M-Spt4/5 elongation complex; F, arrested complex; G, Pol ll-TFIIS reactivation intermediate). Side views are depicted except for (A), which shows the front view (Cramer et al., 2001).
relaxation of the bridge helix allows the incoming template base to enter the active site, completing the NAC. A second NAC cycle in the movie shows the effect of the mushroom toxin a-amanitin, which inhibits Pol II elongation by preventing translocation (Brueckner and Cramer, 2008; Kaplan et al., 2008).
Obstacles and Reactivation
Certain DNA sequences, nucleotide misincorporation, or damaged template DNA form obstacles for NAC progression. Such obstacles induce offline states, which require reactivation before the NAC can resume. The movie illustrates how a frequent obstacle, nucleotide misincorporation, can be overcome by RNA proofreading. A purine-purine mismatch in the active site triggers Pol II pausing, which can also be induced by transcription of a weak DNA-RNA hybrid. The mismatched 3' nucleotide frays away from the DNA template such that it stacks onto the "gating tyrosine" residue Y769 of the largest Pol II subunit (Cheung and Cramer, 2011; Sydowet al., 2009). Pol II backtracking by a single step is followed by endonucleolytic cleavage of a dinucleotide that contains the mismatched nucleotide, reactivating the EC. RNA cleavage is catalyzed by an intrinsic hydrolytic activity of the tunable Pol II active site (Kettenberger et al., 2003) and is stimulated by the RNA 3' end (Zenkin et al., 2006). Cleavage restores a 3'-OH group in the active site, from which elongation resumes.
The movie then shows backtracking and arrest, which can occur upon attempts to elongate through specific DNA sequences (Nudler et al., 1997) or a nucleosome (Kireeva et al., 2005). During backtracking, RNA is extruded into the pore beneath the Pol II active site and is trapped in an RNA-binding site, which prevents forward translocation and NTP binding and results in arrest (Cheung and Cramer, 2011) (Figures 1F and 2F). Reactivation of arrested Pol II requires the elongation factor TFIIS, which stimulates RNA cleavage at the Pol II active site and is animated in two stages. First, domain II of TFIIS binds at the polymerase funnel and causes a conformational change in the polymerase that repositions the backtracked RNA (Cheung and Cramer, 2011). Second, domain III of TFIIS inserts into the pore next to backtracked RNA and reaches the active site with a hairpin that contains three charged residues that stimulate RNA cleavage (Figures 1G and 2G), generating a new RNA 3' end from which transcription resumes.
Current Limitations
The movie reveals gaps in our understanding of the transcription mechanism and encourages a continuation of the current structure-function analysis of Pol II. It defines new open questions about initiation and elongation. For instance, what is the mechanism of DNA melting during the closed-to-open promoter complex transition? It is known that DNA opening requires
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downstream action of TFIIE and TFIIH (Dvir et al., 2001; Kim et al., 2000), and it also involves TFIIB (Knutson and Hahn, 2011; Kostrewa et al., 2009). However, the structural mechanisms remain unclear, although they were recently elucidated for prokaryotic transcription (Feklistov and Darst, 2011). During the closed-to-open transition, some contacts between TFIIB and the polymerase are broken, resulting in movements of the upstream DNA (Treutlein et al., 2012). However, more structural restraints are required before this can be animated and included in the movie. After DNA opening, Pol II scans the template to locate the transcription start site, but the scanning mechanism is poorly understood, and it is not included in the movie. There is also no structural information for the initiating complex, which contains the first two NTP substrates prior to formation of the first phosphodiester bond. Furthermore, where exactly do other initiation factors and coactivators bind, including TFIID and the Mediator?
With respect to elongation, the animated NAC is simplified, as it does not take into account the sampling of NTPs in the active center when Pol II selects the correct substrate. Also, the animation of translocation does not take into account the real nature of the Brownian ratchet formed by the bridge helix and trigger loop. The NAC may be modified as additional conformational states of the EC are observed. For example, a recent structure of a bacterial RNA polymerase with a bound inhibitory protein revealed a new conformation of the enzyme that may be relevant for translocation (Tagami et al., 2010). Also unknown is the structure of the product complex, which contains the newly formed RNA 3' end in the pretranslocation position and the pyrophosphate ion still bound to Pol II. This state was modeled for the movie, assuming a closed trigger loop. Only two elongation factors, Spt4/5 and TFIIS, have been located on Pol II and included in the movie, but structural details for how the many other elongation factors interact with Pol II are lacking. What are the mechanisms used by elongation factors that influence chromatin structure, such as the Paf1 complex, Spt6, or the FACT (facilitates chromatin transcription) complex? In addition, the structural mechanism of Pol II termination remains enigmatic and could not be included in the movie.
Conclusions and Future Directions
Here, we used structural information to generate a molecular movie that captures key aspects of RNA polymerase II initiation and elongation. In the future, the movie may be extended to describe transcription in its cellular context. For example, the mechanisms underlying the coordination of transcription with mRNA processing are partially understood and may be included in the movie when some remaining gaps in our knowledge are closed. This coordination relies on the C-terminal repeat domain (CTD) of Pol II, which extends from the enzyme like a long, flexible tail. The CTD is subject to posttranslational modifications and recruits many different factors at various stages of the transcription cycle. Other flexible elements, including the nascent RNA and histone tails of transcribed nucleosomes, participate in additional interactions with various factors and contribute to the coordination of transcription with other nuclear events. The intricate network of transient and cooperative interactions between these flexible elements and their target factors should
eventually be understood at a level that allows for their animation. As a first step toward this goal, the movie here summarizes a wealth of structural information on the core transcription machinery and provides a useful tool for teaching the mechanism of transcription.
SUPPLEMENTAL INFORMATION
Supplemental Information includes a movie and can be found with this article online at doi:10.1016/j.cell.2012.06.006.
ACKNOWLEDGMENTS
P.C. was supported by the Deutsche Forschungsgemeinschaft, SFB646, TR5, FORI 068, SFB960, NIM, the European Molecular Biology Organization (EMBO), an Advanced Investigator Grant of the European Research Council, LMU excellent research professorship "Molecular systems biology of gene regulation," the LMUinnovativ project Bioimaging Network (BIN), and the Jung-Stiftung.
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