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Structure of the complete elongation complex of RNA polymerase II with basal factors

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Science  01 Sep 2017:
Vol. 357, Issue 6354, pp. 921-924
DOI: 10.1126/science.aan8552

Transcription machinery remains steadfast

Eukaryotic transcription of mRNA is a multistep process mediated by RNA polymerase II (Pol II). Pol II combines with several other factors to form an elongation complex that promotes transcription elongation. Ehara et al. determined the high-resolution structure of the elongation complex by means of x-ray crystallography and cryo-electron microscopy (see the Perspective by Fouqueau and Werner). Multiple elongation factors are distributed on a wide surface of Pol II and establish an RNA exit path and DNA entry or exit tunnels, which facilitate nascent transcript transfer and DNA unwinding or rewinding. The Pol II elongation complex thus adopts a stable architecture suitable for processive transcription.

Science, this issue p. 921; see also p. 871

Abstract

In the early stage of transcription, eukaryotic RNA polymerase II (Pol II) exchanges initiation factors with elongation factors to form an elongation complex for processive transcription. Here we report the structure of the Pol II elongation complex bound with the basal elongation factors Spt4/5, Elf1, and TFIIS. Spt4/5 (the Spt4/Spt5 complex) and Elf1 modify a wide area of the Pol II surface. Elf1 bridges the Pol II central cleft, completing a “DNA entry tunnel” for downstream DNA. Spt4 and the Spt5 NGN and KOW1 domains encircle the upstream DNA, constituting a “DNA exit tunnel.” The Spt5 KOW4 and KOW5 domains augment the “RNA exit tunnel,” directing the exiting nascent RNA. Thus, the elongation complex establishes a completely different transcription and regulation platform from that of the initiation complexes.

Transcription is accomplished by DNA-dependent RNA polymerase (RNAP) through a multistep process consisting of initiation, elongation, and termination. In the initiation of eukaryotic mRNA transcription, RNA polymerase II (Pol II) is associated with general transcription factors (GTFs), forming a huge initiation complex (IC) on the DNA (13). After the synthesis of a certain length of RNA, the GTFs are replaced with elongation factors (EFs). Thus, the IC isomerizes into a processive elongation complex (EC), which serves as a platform for regulation and various transcription-coupled events, such as mRNA processing, chromatin remodeling, and DNA repair (47). Although recent advances in cryo–electron microscopy (cryo-EM) have revealed several IC structures (13), the architecture of the EC still remained elusive. Here we elucidated the Pol II EC structure, including the conserved basal EFs Spt4/5, Elf1, and TFIIS, by using an integrated approach combining x-ray crystallography and cryo-EM.

Spt4/5, a heterodimeric complex of Spt4 and Spt5 (NusG in bacteria), is implicated in processive transcription elongation (8, 9), promoter-proximal pausing in higher eukaryotes (as DRB sensitivity-inducing factor) (8, 10), and many transcription-coupled events (47). Spt5 is a multidomain protein composed of the conserved NGN (NusG N-terminal) domain, which binds Spt4, and different numbers of KOW (Kyprides-Onzonis-Woese) domains, which are connected by flexible linkers and the C-terminal repeat region (CTR) (Fig. 1A) (1113). Multiple KOW domains specific to eukaryotic Spt5 are crucial for the establishment of the EC and Spt4/5 functions (14, 15), but their locations on Pol II and functions in transcription have remained enigmatic. To assess which KOW domain is essential for Pol II binding and transcription, we created a series of Spt4/5 variants and investigated their transcription-stimulation functions for Pol II from the yeast Komagataella pastoris (16) (Fig. 1, A and B). Surprisingly, only KOW5 is necessary and sufficient for the transcription stimulation, whereas the other KOW domains, the NGN domain, and Spt4 were dispensable. These data underscore the importance of KOW5 in the Spt4/5 function as an EF.

Fig. 1 Crystal structure of Pol II EC with Spt5 KOW5 and Elf1.

(A) The Spt4/5 and Elf1 constructs used in this study (numbers represent amino acid residue positions). The regions studied in the crystallographic and cryo-EM analyses are indicated by the red and green boxes, respectively. (B) Polyacrylamide gel electrophoresis analysis of in vitro transcription stimulation by Spt4/5 variants. (C) Overall structure of the Pol II EC–KOW5-Elf1 complex. (D) Close-up views of the Spt5 KOW5–binding site in two different orientations. (E) The Elf1-binding site and the DNA entry tunnel, shown in two different orientations. In the right panel, the N-terminal basic tail of Elf1 was presumed and is depicted as a dashed line. (F) The amino acid sequence of the Elf1 N-terminal tail, in which the basic residues are highlighted. β1 represents the first β strand of Elf1.

Elongation factor 1 (Elf1 in yeast; ELOF1 in human) is a small protein conserved in eukaryotes and several archaeal classes, including Crenarchaeota (fig. S1) (17). Elf1 displays synthetic lethality with several EFs, including Spt4, Spt5, and TFIIS in yeast (18). Chromatin immunoprecipitation analyses revealed that Elf1 accompanies transcribing Pol II in vivo (18, 19). As Elf1 slightly delays transcription elongation by K. pastoris Pol II in vitro, it seems to directly interact with Pol II and function in transcription regulation (fig. S2).

To understand how the Spt5 KOW5 domain and Elf1 associate with Pol II, we solved the cocrystal structure of the K. pastoris Pol II EC bound with KOW5 and Elf1 at 3.0 Å resolution (Fig. 1C, fig. S3, and table S1). KOW5 binds in a complementary pocket formed at the junction of five Pol II subunits: Rpb1, Rpb2, Rpb3, Rpb11, and Rpb12 (Fig. 1D), which interact extensively through electrostatic and polar interactions between well-conserved amino acid residues (fig. S4). Notably, KOW5 bridges the Rpb1 dock domain and the Rpb2 wall domain of Pol II, becoming an integral part of the “RNA exit tunnel,” which is described later in the text. The location of KOW5 constrains the relative orientations of the shelf and core modules (20) of Pol II. Thus, KOW5 also seems to adjust the Pol II conformation (21) so it is suitable for processive elongation.

Elf1 is located between the Rpb2 lobe domain and the Rpb1 clamp-head domain of Pol II (Fig. 1E). While the α helix (the Pol II–binding helix) of Elf1 interacts with the Arg-rich face at the tip of the Rpb2 lobe (fig. S5), the opposite side of Elf1 reaches the Rpb1 clamp head. As a consequence, Elf1 fills the gap between the core and clamp modules over the central cleft of Pol II and thus completes a closed “DNA entry tunnel” for the passage of the downstream DNA duplex. Although the N-terminal basic tail is disordered, it should interact with the DNA held in the closed tunnel (Fig. 1, E and F). Even though Elf1 delays transcription in vitro, it may stabilize the EC by preventing DNA dissociation. Consistently, the effect of Elf1 was compromised when the N-terminal tail was deleted or the Pol II–interacting residues were mutated (fig. S2).

Although we identified the binding site of Spt5 KOW5 on Pol II, the positions and nucleic acid interactions of the other KOW domains, the NGN domain, and Spt4 were still unclear. To address these problems, we performed cryo-EM single-particle analyses of the Pol II EC bound with Spt4/5, including an Spt5 variant (residues 217 to 815, from NGN to KOW5) and an inactive variant of TFIIS (table S2). The three-dimensional reconstruction with 682,749 particles yielded the Pol II EC structure at an overall resolution of 3.8 Å (fig. S6). Structural heterogeneities were observed around the upstream DNA, the Pol II Rpb4/7 stalk, and the secondary channel. Focused classifications for these regions successfully resolved the upstream DNA-bound Spt4 and Spt5 NGN-KOW1, the stalk-bound Spt5 KOW4, and the secondary channel–bound TFIIS, respectively (figs. S7 to S11).

In the cryo-EM structure, the Spt5 NGN domain is docked to the tip of the Rpb1 clamp coiled coil and occupies the space between the clamp (Rpb1 coiled coil) and core (Rpb2 protrusion and lobe) modules (Fig. 2, A and B). The closed clamp coincides with the fluorescence resonance energy transfer study on an archaeal RNAP (22), and the NGN position is generally consistent with that seen in the partial x-ray and 13 Å cryo-EM structures of the archaeal RNAP and the 26 Å negative-stain EM structure of mammalian Pol II (2325). KOW1 is bound to the “zipper” loop of the Rpb1 clamp (Fig. 2, C and D). In addition, the eukaryote-specific insertion of KOW1 contacts the tip of the Rpb2 wall. Thus, KOW1 bridges the clamp and core modules of Pol II on the other side of the NGN domain. Spt4 is tightly associated with the NGN and resides between KOW1 and the Rpb2 protrusion.

Fig. 2 Cryo-EM structure of Pol II EC with Spt4/5 and TFIIS.

Overall structure of the complex showing (A) the “DNA-binding” face and (B) the “RNA-exit” face. (C) Interactions of Spt4 and Spt5 NGN-KOW1 with Pol II and DNA. The cryo-EM map (before sharpening) corresponding to Spt4, Spt5 NGN-KOW1, and the upstream DNA is overlaid. (D) Top view of the DNA exit tunnel. (E) Side view of the DNA exit tunnel. (F) The same view as in (E) with the surface electrostatic potentials of Spt4 and Spt5 NGN-KOW1. (G) Close-up view of the RNA exit site. The cryo-EM map (before sharpening) corresponding to the exiting RNA and Spt5 KOW4-KOW5 is overlaid. The KOW4-KOW5 linker and a possible path of the exiting RNA are depicted by a green line and a red dashed arrow, respectively. (H) Surface electrostatic potentials of the Spt5 KOW4-KOW5 domains, the Rpb1 dock, and the Rpb4/7 stalk.

As a consequence, the Spt5 NGN-KOW1 domains and Spt4, together with the Rpb2 protrusion and wall, establish a “DNA exit tunnel” that covers approximately one helical pitch of the upstream DNA duplex (Fig. 2, C and E). The conserved Arg232, Lys235, Lys237, Arg241, and Lys269 residues of the NGN, together with Lys416, Arg419, Arg423, Arg427, Arg433, Lys442, and Lys463 of the Rpb2 protrusion, form a positively charged surface, which contacts the proximal five base pairs of the upstream DNA duplex (positions –14 to –10) (Fig. 2F and fig. S12). In addition, three loops of the NGN (residues 230 to 235, 265 to 270, and 286 to 296) contact the single-stranded part of the nontemplate DNA (positions –5 to –9) in the transcription bubble, consistent with the previous cross-linking study (14). KOW1 also provides a surface rich in basic and polar residues (Lys329, Asn330, Lys334, Lys386, Lys428, Arg431, Gln433, and Asn434), through which it interacts with the distal part of the upstream DNA duplex (positions –17 to –12). The β1-β2 loop of KOW1 (residues 328 to 335) fits in the minor groove of the DNA (fig. S13).

Spt4 and the eukaryote-specific insertion of KOW1 loosely surround the distal part of the DNA. Although they are farther apart from the DNA, they contain several basic residues that could potentially participate in DNA binding. Thus, the DNA exit tunnel assumes a funnel-like shape, which allows a certain degree of DNA bending in its distal part while restricting the DNA orientation in the proximal part. The focused classification for the upstream DNA depicted several KOW1-lacking classes with different DNA and NGN orientations (fig. S14). This emphasizes the importance of KOW1 for defining the appropriate DNA orientation and explains why the deletion of KOW1 is lethal in yeast (14).

Spt5 KOW4 is bound to the oligonucleotide/oligosaccharide-binding (OB) fold domain of the stalk subunit Rpb7 (Fig. 2B and fig. S15). The location of KOW4 is in good agreement with the previous cross-linking study (7). Thus, Spt5 KOW1, KOW4, KOW5, and Rpb7 are distributed around the rim of the RNA exit tunnel, expanding it into a large funnel-like structure. Although the tandemly packed KOW2 and KOW3 domains (12) were not resolved, they should reside between KOW1 and KOW4, possibly on the Rpb1 clamp according to the cross-linking study (7), and could participate in the expanded RNA exit tunnel.

The density for the exiting RNA is observed along a channel composed of the Spt5 KOW5, the Rpb1 dock, and the Rpb2 wall (Fig. 2G). This view is consistent with the observations that KOW5 is cross-linked to nascent RNA (15) and important for transcription processivity (14). As KOW5 blocks one of the two previously proposed RNA exit paths (path 2) (20, 26), the RNA should be directed through the remaining path 1 toward the Pol II stalk. A weak density for the linker connecting KOW4 and KOW5 was observed, which also participates in the path. A long stretch of nascent RNA is required for the stable binding of Spt4/5 to the Pol II EC (27), and the RNA interacts with the stalk subunit Rpb7 (26). KOW4 cooperates with Rpb7 to form a basic patch (Fig. 2H), which could serve as the RNA-binding site. Thus, the Spt5 KOWs augment the RNA exit tunnel, likely supporting the efficient exit of the nascent RNA.

The combination of the present x-ray and cryo-EM structures provides a reliable composite model of the Pol II EC, including the basal EFs Spt4/5, Elf1, and TFIIS (Fig. 3 and movie S1). The EFs are distributed on a wide area of the Pol II surface and constitute or expand the DNA entry tunnel (Elf1), the DNA exit tunnel (Spt4 and Spt5 NGN-KOW1), and the RNA exit tunnel (Spt5 KOW1 to 5). Spt5 NGN and Elf1 are adjacent to each other at the inner corner of the 90°-bent DNA. Spt4/5 and Elf1 achieve almost symmetrical coverage of the upstream and downstream parts of the DNA, encompassing it from positions –15 to +16 (Fig. 3E). The established DNA entry and exit tunnels enable proper DNA unwinding and rewinding in the defined size of the transcription bubble. Spt5 KOW1, possibly with the unresolved KOW2 and KOW3, physically separates the DNA and RNA exit faces, preventing potential DNA-RNA interactions such as R-loop formation. These features extensively represent the architecture required for processive transcription, while counteracting pausing, backtracking, arrest, and premature termination. The KOW5 location suggests that the CTR, the intrinsically unstructured extension of Spt5, protrudes from the rim of the RNA exit tunnel (Fig. 3D). This is quite reasonable, as the CTR, as well as the Pol II C-terminal domain (CTD), recruits various factors, including mRNA-processing machineries (46, 28, 29).

Fig. 3 The architecture of the Pol II EC.

(A) The DNA and RNA binding sites in the EC (the EFs are omitted). (B) The same as (A) but with bound Spt4/5, Elf1, and TFIIS (the composite model showing the DNA entry and exit tunnels). (C) The view of the RNA exit tunnel (the EFs are omitted). (D) The same as (C) but with the EFs (the expanded RNA exit tunnel). The KOW4-KOW5 linker, Pol II CTD, and Spt5 CTR are depicted by green, brown, and violet lines, respectively. (E) Schematic diagram summarizing the interactions between nucleic acids and EFs.

A structural comparison reveals that many components of the EC are mutually exclusive with those of ICs (13) (fig. S16). Spt4, Spt5 NGN, and Elf1, which seal the Pol II cleft, overlap with the DNA duplex in the preinitiation complex. Moreover, Spt4 and Spt5 KOW4 in the EC overlap with the RAP30 subunit of TFIIF and the α subunit of TFIIE, respectively, in ICs. These observations indicate that the EC is only achieved after the isomerization from IC to EC and the exchange of GTFs with EFs. Consistently, the exchange of archaeal TF(II)E and Spt4/5 was reported (13, 30). Collectively, the EC establishes a different architecture from those of ICs, for processive transcription, regulation, and many transcription-coupled events.

Supplementary Materials

www.sciencemag.org/content/357/6354/921/suppl/DC1

Materials and Methods

Figs. S1 to S16

Tables S1 and S2

References (3145)

Movie S1

References and Notes

  1. Acknowledgments: We thank Y. Tomabechi, T. Uchikubo-Kamo, and T. Osanai for technical assistance. The synchrotron radiation experiments were performed at BL41XU of SPring-8 with the approval of the Japan Synchrotron Radiation Research Institute (proposal nos. 2014B1265, 2015B2040, 2016A2526, and 2016B2526) and at NE3A of the Photon Factory with the approval of the Photon Factory Program Advisory Committee (proposal no. 2015G520). We thank H. Okumura and K. Hasegawa for assistance with data collection at SPring-8. This work was supported in part by the Japan Society for the Promotion of Science KAKENHI grant numbers JP15H04344 (to S.S.) and JP15H01656 (to H.S.), the Platform Project for Supporting Drug Discovery and Life Science Research funded by the Japan Agency for Medical Research and Development, and the RIKEN Pioneering Project, Dynamic Structural Biology. The atomic coordinates and structure factors for the EC bound with Elf1 and Spt5 KOW5 have been deposited in the Protein Data Bank (PDB ID 5XOG). The cryo-EM density map and the atomic coordinates for the EC bound with Spt4/5 and TFIIS were deposited in the Electron Microscopy (EM) Data Bank (accession code EMD-6747) and the Protein Data Bank (PDB ID 5XON), respectively.
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