Structural Basis of Transcription Initiation by RNA Polymerase II

Structural basis of transcription initiation by RNA polymerase II

Sarah Sainsbury*, Carrie Bernecky* and Patrick Cramer Abstract | Transcription of eukaryotic protein-coding genes commences with the assembly of a conserved initiation complex, which consists of RNA polymerase II (Pol II) and the general transcription factors, at promoter DNA. After two decades of research, the structural basis of transcription initiation is emerging. Crystal structures of many components of the initiation complex have been resolved, and structural information on Pol II complexes with general transcription factors has recently been obtained. Although mechanistic details await elucidation, available data outline how Pol II cooperates with the general transcription factors to bind to and open promoter DNA, and how Pol II directs RNA synthesis and escapes from the promoter.

Transcription of the eukaryotic genome is carried In the classical model, a Pol II–TFIIF complex binds out by nuclear RNA polymerase I (Pol I), Pol II and to a pre-formed TFIIB–TBP–DNA promoter complex, Pol III. Whereas Pol I transcribes the rRNA precur- resulting in the formation of a core initiation complex sor, Pol III transcribes small non-coding RNAs such as (FIG. 1). The core initiation complex is conserved in the tRNAs. Pol II is a 12‑subunit enzyme that transcribes Pol I and Pol III transcription systems, which also use TBP protein-coding genes to produce mRNAs. Pol II regu- and contain proteins with homologies to TFIIB and TFIIF lation underlies cell differentiation, the maintenance of (reviewed in REF. 7). The core initiation complex binds to cell identity and the responses of cells to environmen- TFIIE and TFIIH to form a complete PIC that contains tal changes. It occurs at different stages of transcrip- closed, double-stranded promoter DNA (TABLE 1). In the tion, although regulation at the stage of initiation is presence of nucleoside triphosphates, a central DNA a key mechanism for the control of gene expression. region is melted, leading to a ‘transcription bubble’ and Understanding Pol II regulation, therefore, requires the formation of the open promoter complex. In the open detailed insights into the structure of the Pol II ini- promoter complex, the DNA template strand passes near tiation complex and the molecular mechanisms of the Pol II active site and can programme DNA-templated transcription initiation. RNA chain synthesis. Most general transcription fac- For initiation, Pol II assembles with the general tors are modular and contain structured domains that transcription factors TFIIB, TFIID, TFIIE, TFIIF are connected by flexible linkers. Upon assembly of the and TFIIH, which are collectively known as the gen- PIC, these factors adopt their functional structure. Their eral transcription factors, at promoter DNA to form linker regions fold on the Pol II surface, and their protein the pre-initiation complex (PIC) (TABLE 1). According domains locate to sites where they can exhibit specialized to exemplary studies with a subset of promoters, the functions. Detailed structural information on how gen- Max Planck Institute for general transcription factors cooperate with Pol II to eral factors interact with Pol II is scarce, but recent studies Biophysical Chemistry, bind to and open promoter DNA, and to initiate RNA have increased our understanding of the 3D architecture Department of Molecular synthesis and stimulate the escape of Pol II from the of initiation complexes8–13. Biology, Am Fassberg 11, 37077 Göttingen, Germany. promoter. TFIID contains the TATA box-binding pro- In this Review, we summarize known structural *These authors contributed tein (TBP) and several TBP-associated factors (TAFs). information on Pol II initiation complexes and discuss equally to this work. Whereas TBP is required for transcription from all the functions of PIC components. We describe available Correspondence to P.C. promoters, the TAFs have promoter-specific func- structures for the general transcription factors and their e-mail: patrick.cramer@ tions. Order‑of‑addition experiments1 combined with complexes (TABLE 2; see Supplementary information S1 mpibpc.mpg.de doi:10.1038/nrm3952 in vivo analysis led to the classical model of stepwise PIC (table)), following the order of the stepwise assembly Published online assembly (reviewed in REFS 2–6), although alternative model of PIC formation. We start with the recruitment 18 February 2015 assembly pathways are possible. of initiation factors to promoter DNA, the formation of

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Table 1 | Subunits of Pol II and general transcription factors Factor Gene name Mass (kDa) Uniprot accession number Copies Yeast Human Yeast Human Yeast Human Pol II (RNAP*): transcribing enzyme RPB1 RPO21 POLR2A 191.6 217.2 P04050 P24928 1 RPB2 RPB2 POLR2B 138.8 133.9 P08518 P30876 1 RPB3 RPB3 POLR2C 35.3 31.4 P16370 P19387 1 RPB4 RPB4 POLR2D 25.4 16.3 P20433 O15514 1 RPB5‡ RPB5 POLR2E 25.1 24.6 P20434 P19388 1 RPB6‡ RPO26 POLR2F 17.9 14.5 P20435 P61218 1 RPB7 RPB7 POLR2G 19.1 19.3 P34087 P62487 1 RPB8‡ RPB8 POLR2H 16.5 17.1 P20436 P52434 1 RPB9 RPB9 POLR2I 14.3 14.5 P27999 P36954 1 RPB10‡ RPB10 POLR2L 8.3 7.6 P22139 P62875 1 RPB11 RPB11 POLR2J 13.6 13.3 P38902 P52435 1 RPB12‡ RPB12 POLR2K 7.7 7.0 P40422 P53803 1 Total 513.6 516.7 (12 subunits) TFIIA§: TBP stabilization and counteracts repressive effects of negative co‑factors Large subunit TOA1 GTF2A1 32.2 41.5 P32773 P52655 1 Small subunit TOA2 GTF2A2 13.5 12.5 P32774 P52657 1 Total 45.7 54.0 (2 subunits) TFIIB: Pol II recruitment, TBP binding and TSS selection TFIIB (TFB*) SUA7 GTF2B 38.2 34.8 P29055 Q00403 1 TFIID: Pol II recruitment and promoter recognition TBP (TBP*): recognition TBP TBP 27.0 37.7 P13393 P20226 1 of the TATA box TAF1 TAF1 TAF1 120.7 212.7 P46677 P21675 1 TAF2 TAF2 TAF2 161.5 137.0 P23255 Q6P1X5 1 TAF3 TAF3 TAF3 40.3 103.6 Q12297 Q5VWG9 1 TAF4 TAF4 TAF4 42.3 110.1 P50105 O00268 2 TAF5 TAF5 TAF5 89.0 86.8 P38129 Q15542 2 TAF6 TAF6 TAF6 57.9 72.7 P53040 P49848 2 TAF7 TAF7 TAF7 67.6 40.3 Q05021 Q15545 1 TAF8 TAF8 TAF8 58.0 34.3 Q03750 Q7Z7C8 1 TAF9 TAF9 TAF9 17.3 29.0 Q05027 Q16594 2 TAF10 TAF10 TAF10 23.0 21.7 Q12030 Q12962 2 TAF11 TAF11 TAF11 40.6 23.3 Q04226 Q15544 1 TAF12 TAF12 TAF12 61.1 17.9 Q03761 Q16514 2 TAF13 TAF13 TAF13 19.1 14.3 P11747 Q15543 1 TAF14|| TAF14 NA 27.4 NA P35189 NA 3 Total 1,200¶ 1,300¶ (14–15 subunits) TFIIE: recruitment of TFIIH and open DNA stabilization TFIIEα (TFE*) TFA1 GTF2E1 54.7 49.5 P36100 P29083 1 TFIIEβ TFA2 GTF2E2 37.0 33.0 P36145 P29084 1 Total 91.7 82.5 (2 subunits)

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Table 1 (cont.) | Subunits of Pol II and general transcription factors Factor Gene name Mass (kDa) Uniprot accession number Copies Yeast Human Yeast Human Yeast Human TFIIF§: TSS selection and stabilization of TFIIB TFIIFα TFG1 GTF2F1 82.2 58.2 P41895 P35269 1 TFIIFβ TFG2 GTF2F2 46.6 28.4 P41896 P13984 1 TFG3# TAF14 NA 27.4 NA P35189 NA NA Total 156.2 86.6 (2–3 subunits) TFIIH§ (core): promoter opening and DNA repair Subunit 1 (p62) TFB1 GTF2H1 72.9 62.0 P32776 P32780 1 Subunit 2 (p44) SSL1 GTF2H2 52.3 44.4 Q04673 Q13888 1 Subunit 3 (p34) TFB4 GTF2H3 37.5 34.4 Q12004 Q13889 1 Subunit 4 (p52) TFB2 GTF2H4 58.5 52.2 Q02939 Q92759 1 Subunit 5 (p8) TFB5 GTF2H5 8.2 8.1 Q3E7C1 Q6ZYL4 1 XPD subunit: ATPase; RAD3 ERCC2 89.8 86.9 P06839 P18074 1 DNA repair XPB subunit: ATPase; SSL2 ERCC3 95.3 89.3 Q00578 P19447 1 promoter opening Total 414.5 377.3 (7 subunits) TFIIH (kinase module): CTD phosphorylation Cyclin H CCL1 CCNH 45.2 37.6 P37366 P51946 1 CDK7 KIN28 CDK7 35.2 39.0 P06242 P50613 1 MAT1 TFB3 MNAT1 38.1 35.8 Q03290 P51948 1 Total 118.5 112.4 (3 subunits) CTD, C‑terminal domain; NA, not available; Pol, RNA polymerase; TAF, TBP-associated factor; TBP, TATA-box-binding protein; TFIIA, transcription initiation factor IIA; TSS, transcription start site. *Archaeal homologue. ‡Factor shared between Pol I, Pol II and Pol III. §No known archaeal homologue. ||Component of TFIID, TFIIF and chromatin remodelling complexes. ¶Approximate molecular weight. #TFG3 is a component of TFIID, TFIIF and chromatin remodelling complexes; the yeast-specific subunit is non-essential as part of TFIIF and as part of TFIID212.

the core initiation complex and the interaction of this TFIIB was found to be located on the Pol II dock domain complex with the auxiliary factor TFIIA. We then dis- using biochemical probing26 and X‑ray crystallography27 cuss TFIIE and TFIIH and their roles in promoter DNA (BOX 1). As this domain was not present in the TFIIB– opening. Finally, we discuss how recent data on the TBP–DNA complex structure19, a model for the Pol II– structure of TFIID provide insights into its functions in TFIIB–TBP–DNA complex had to be derived with the determining promoter specificity. use of site-specific protein cleavage probing28,29 (BOX 1). In 2009, the structure of the complete 12‑subunit A brief history of initiation complex architecture Pol II bound by TFIIB confirmed the location of the Structural analyses of the PIC started over two decades B‑ribbon domain on the dock and positioned one of ago. Structures of TBP in free form14,15 and bound to two cyclin folds in the carboxy‑terminal B‑core domain DNA16,17 revealed that TBP is a saddle-shaped molecule of TFIIB on the Pol II wall10. This enabled modelling of that binds to the DNA minor groove and bends DNA Pol II–TFIIB–TBP–DNA complexes with closed and by 90 degrees. In later structures, TFIIA and TFIIB were open promoter DNA10. An independent TFIIB structure seen to flank the TBP–DNA complex on either side18–20. bound to the 10‑subunit Pol II core enzyme provided When the yeast Pol II structure became available21–25, similar results11. The crystallographically derived models it revealed many domains that have putative functions also resembled the models that were derived biochemi- during initiation, including the dock, wall and clamp cally28,29. A recently obtained high-resolution crystal domains, and an RNA exit tunnel. The challenge then structure of a Pol II–TFIIB complex (FIG. 2a) additionally was to understand the relative positions of TFIIB and contained DNA and a short RNA transcript, and revealed Pol II, because subsequent superimposition of the known details about the TFIIB ‘B‑reader’ and ‘B‑linker’ regions TFIIB–TBP–DNA complex would reveal the position of that connect the B‑ribbon to the B‑core and run through TBP and promoter DNA on Pol II. The B‑ribbon (also the polymerase active centre cleft30. These models could known as the amino‑terminal zinc ribbon) domain of be extended to the core initiation complex (FIG. 2b) by

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including TFIIF, which was positioned on Pol II with Unbound promoter TAFs the use of protein–protein crosslinking31,32 (BOX 1). TBP Crosslinking of the core initiation complex confirmed the Pol II–TFIIF–TFIIB–TBP–DNA model12. In 2013, the stepwise assembly of a human PIC was visualized by electron microscopy (EM)9. In this study, Promoter DNA TFIIF, TFIIE and TFIIH were sequentially added to TFIIA a complex comprising Pol II, TFIIB, TBP, DNA and TFIIB TFIIA. The architecture of the human PIC showed a remarkable similarity to the yeast core initiation com- Upstream promoter TFIIB (FIG. 2b) plex . Additionally, it revealed locations of TFIIE complex on the polymerase clamp domain that were consistent +1 with the locations of the homologous yeast factor8,33, TFIIA and a downstream location of TFIIH that was also topo- TAFs logically similar to that derived for the yeast complex8 (FIG. 3a). The human PIC architecture derived by EM was Pol II also consistent with that derived by early DNA–protein TFIIF photo-crosslinking experiments that located TFIIH near 34 DNA downstream of the initiation complex . A different Core PIC PIC architecture was suggested by an alternative study that analysed the yeast PIC by EM and located TFIIH Pol II above the Pol II cleft13. Very recently, an alternative study 249 of the yeast initiation complex structure consistent with Upstream DNA 9 the human study was published. TFIIF Downstream DNA TBP and promoter binding TFIIE TFIIH The first step in canonical PIC assembly is the sequence- TFIIH TFIIE specific binding of TBP to the TATA box. The TATA Closed PIC kinase box has a consensus sequence of TATAWAWR35 and is located around 30 bp upstream of the transcription start site (TSS) in humans. There is early evidence that the TFIIH factor that binds to the TATA box is functionally con- core served from yeast to humans36 and that the TBP sub unit of TFIID is sufficient for transcription in vitro37,38 and in vivo39. Although most promoters do not contain ATP canonical TATA boxes (TATA-less promoters), TBP and other general factors were located genome-wide on Open PIC most promoters in yeast40. TBP can bind to and bend variants of TATA DNA41, which suggests that the overall architecture of the initiation complex is similar at TATA‑containing and TATA-less promoters. Transcription bubble Free TBP has a saddle-shaped structure14,15. Structures of TBP bound to the TATA box DNA in yeast17, plant16,42 and human43–45 cells uncovered a highly conserved bind- NTPs ing mode. The concave surface of TBP binds to the minor Initially transcribing groove of the TATA box, inducing a ~90‑degree bend in complex the DNA. Although TBP binds to DNA with nanomolar Nascent RNA affinity16, relatively few base-specific DNA contacts were observed. DNA specificity apparently results from the propensity of the A/T‑rich DNA to form a hydro phobic surface that is complementary to TBP. The abil- ity of TBP to bind to various DNA sequences probably enables it to function in transcription initiation by any Elongation factors of the three eukaryotic RNA polymerases (reviewed in Initiation REF. 7). Investigating how transcription initiates at TATA- factors less promoters remains an important goal for the future. Elongation complex The auxiliary factor TFIIA is not required for basal 5′ cap Elongation factors transcription but can stabilize the TBP–DNA complex46. TFIIA is Pol II‑specific and stimulates constitutive transcription (that is, transcription at basal levels) and activated transcription47. Structures of yeast18,20

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◀ Figure 1 | Schematic of Pol II transcription initiation. Depicted is the canonical TFIIB and Pol II recruitment model for stepwise pre-initiation complex (PIC) assembly from general transcription TFIIB is required for Pol II recruitment to the pro- factors (various colours) and RNA polymerase II (Pol II; grey) on promoter DNA. The names moter50–53 and facilitates TBP binding to DNA and DNA for the intermediate complexes that form during the initiation–elongation transition are bending54. The functions of TFIIB in the recruitment provided to the left of the images. TFIID or its TATA box-binding protein (TBP) subunit binds to promoter DNA, inducing a bend. The TBP–DNA complex is then stabilized by of Pol II and in the interaction of TBP with promoter TFIIB and TFIIA, which flank TBP on both sides. The resulting upstream promoter complex DNA are due to its N- and C‑terminal domains, respec- 55–57 is joined by the Pol II–TFIIF complex, leading to the formation of the core PIC. Subsequent tively . Structural studies revealed an N‑terminal binding of TFIIE and TFIIH complete the PIC. In the presence of ATP, the DNA is opened B‑ribbon domain58,59 and a C‑terminal B‑core region, (forming the ‘transcription bubble’) and RNA synthesis commences. Finally, dissociation which contains two cyclin domains that bind to TBP and of initiation factors enables the formation of the Pol II elongation complex, which is upstream DNA19,60. In the archaeal transcription system, associated with transcription elongation factors (blue). NTP, nucleoside triphosphate; TBP and the TFIIB homologue, TFB, are the only required TAF, TBP-associated factor. initiation factors61, and the structure of an archaeal TBP– TFB–DNA complex resembles that of its eukaryotic counterpart62. TFIIB interacts with the flanking regions and human48 TFIIA–TBP–DNA complexes revealed a of the TATA box to bind to upstream and downstream boot-shaped TFIIA heterodimer that does not alter the DNA sequences that can contain TFIIB recognition TBP–DNA structure (see figure 3a in REF. 18). TFIIA is elements (from here on referred to as B recognition ele- composed of two conserved domains, a 4‑helix bundle ments (BREs))63–65. Recognition of BREs can explain and a 12‑stranded β-barrel that binds to the upstream oriented assembly of the initiation complex despite the region of the TATA box and the underside of the TBP pseudo‑symmetric nature of the TBP–DNA complex66–68. saddle, which explains how TFIIA stabilizes the TBP– Structures of Pol II–TFIIB complexes showed how the DNA complex. Consistent with this, the stability of the B‑ribbon of TFIIB binds to the dock domain of Pol II to TFIIA–TBP–DNA complex depends on the sequence of recruit the polymerase27 and later showed that the first the DNA that is bound49. cyclin domain in the B‑core region is located on the wall

Table 2 | Structural information on general transcription factors Factor Complex or subunits Method PDB or EMDB codes Refs TFIIA TFIIA–TBP–DNA complex X‑ray 1YTF; 1NH2; 1NVP; 1RM1 20,48 TFIIB TFIIB domains X‑ray 3H4C 213 NMR 1PFT; 1TFB; 1DL6; 1RLY; 1RO4; 2PHG 58,59,214–216 TFIIB core–TBP–DNA complex X‑ray 1VOL; 1AIS; 1D3U; 1C9B 19,62,66,68 Pol II–TFIIB complexes X‑ray 1R5U; 3K1F; 3K7A; 4BBR; 4BBS 10,11,27,30 TFIIF TFIIFα and/or TFIIFβ X‑ray 3NFG; 3NFF; 1F3U; 1I27; 1J2X 96,98,132,217 NMR 1ONV; 1NHA; 2K7L; 2BBY 97,101,218,219 TFIIE TFIIEα and/or TFIIEβ X‑ray 1Q1H; 3NFH; 3NFI 132,133 NMR 2JTX; 2RNR; 1VD4; 1D8J; 1D8K 134–136,169 TFIIH TFIIH core subunits or domains X‑ray 1YDL; 3DOM; 3DGP; 2VSF; 4ERN 220–222 NMR 1PFJ; 1Y5O; 1Z60; 2DII; 2GS0; 2JNJ; 2K2U; 2L2I; 2LOX; 2M14; 223–231 2MKR TFIIH kinase module subunits or X‑ray 1JKW; 1UA2 157,159 domains NMR 1G25 160 TFIIH complex EM EMD‑2309 9 TFIIE–TFIIH Complex of domains of TFIIEα and NMR 2RNQ 135 TFIIH subunit 1 (p62) TFIID TAF domains X‑ray 1TAF; 1BH8; 1BH9; 1EQF; 1H3O; 2NXP; 2J49; 2J4B; 2P6V; 196,204,232–241 3AAD; 3HMH; 3UV4; 3UV5; 3QRL; 4ATG; 4B0A; 4OY2 NMR 1TBA; 2K16; 2K17; 2L7E 195,242,243 TFIID and large TAF complexes EM EMD‑1194; EMD‑1195; EMD‑1196; EMD‑5026; EMD‑5135; 191,208,209, EMD‑5134; EMD‑5175; EMD‑2229; EMD‑2230; EMD‑2231; 211,244,245 EMD‑2287; EMD‑2284 TFIID with additional factors EM EMD‑5176; EMD‑5178; EMD‑5177; EMD‑2283; EMD‑2282 191,208 Pol II Complexes of Pol II with various EM EMD‑5343; EMD‑5344; EMD‑5407; EMD‑2304; EMD‑2305; 9,13,246–248 complexes initiation factors EMD‑2306; EMD‑2307; EMD‑2394; EMD‑2308 EM, electron microscopy; EMDB, Electron Microscopy Data Bank; NMR, nuclear magnetic resonance; PDB, Protein Data Bank; Pol II, RNA polymerase II; TAF, TBP-associated factor; TBP, TATA box-binding protein. Owing to the large number of structures for free TBP, for TBP only structures including additional general transcription factors are included. Structures that are not available in the RCSB PDB or the EMDB are omitted.

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Box 1 | Experimental methods used to elucidate the Pol II initiation complex structure Site-specific protein cleavage probing Chemical probes are incorporated at specific sites of a protein within a protein complex and buffer conditions are changed such that these probes induce the cleavage of nearby protein backbone chains. If the structure of the nearby proteins is known, the approximate position of a protein containing the probes on the surface of a protein complex can be derived. X‑ray crystallography The protein complex of interest is purified and crystallized. The crystal structure is then determined using synchrotron radiation and X‑ray diffraction. X‑ray crystallography normally results in highly reliable 3D atomic structures. The drawbacks of this method are that a large quantity of protein must be prepared and that often crystals of sufficient quality cannot be obtained. Cryo-electron microscopy The purified protein complex is suspended in a thin layer of liquid on a grid, flash-frozen and imaged under the electron microscope. The obtained images of thousands of single particles are aligned in the computer, and the 3D structure is calculated from back-projection by combining images from different views after determining their relative orientations. The advantage of this method is that a lower amount of material is required compared to X‑ray crystallography. The disadvantage is that often the resolution of this method is insufficient to derive molecular mechanistic insights, although topological insights can generally be obtained. Recent advances in detector technology have led to a dramatic increase in resolution, and detailed insights are expected from electron microscopy in the future. Crosslinking and mass spectrometry The protein complex of interest is incubated with a bifunctional chemical crosslinking reagent that covalently links surface lysine residues. After protease digestion, the resulting peptides are analysed by mass spectrometry and sites of crosslinking are identified by analysis of the obtained data. Interprotein crosslinks can then be used to verify the complex models obtained after fitting available crystal structures to cryo-electron microscopic maps.

of Pol II to position DNA over the Pol II active centre site residues and stabilizing a closed polymerase clamp30. cleft10,11. By combining the crystal structures of the Pol II– Fourth, TFIIB stabilizes an early initiation complex con- TFIIB complexes with the structure of the TFIIB–TBP– taining a five-nucleotide RNA strand27, and the B‑reader DNA complex, models of closed and open promoter loop blocks the path of the RNA beyond six nucleotides complexes containing TFIIB, TBP, DNA and Pol II were and may aid DNA–RNA strand separation by directing obtained10,11 (FIG. 3b) that closely resemble the models the RNA to the exit tunnel30. Last, TFIIB is released from derived biochemically28 and that explain how TBP and the complex when the RNA reaches a length of 12–13 TFIIB cooperate to load promoter DNA onto Pol II. nucleotides76 and clashes with the B-ribbon30. The interplay between TFIIB and nucleic acids is critical for the Post-recruitment functions of TFIIB. The Pol II–TFIIB initiation-to-elongation transition77. In contrast to human structures also revealed that the TFIIB region connect- Pol II, which initiates transcription around 30 bp down- ing the B‑ribbon and B‑core domains traverses the Pol II stream of the TATA box, yeast Pol II scans DNA for a cleft10,11 to form two distinct elements, the B‑reader site of RNA chain initiation further downstream73, but the and B-linker10. Following the N‑terminal B‑ribbon, the mechanistic basis for this difference remains unknown. polypeptide chain runs along the polymerase RNA exit channel in the opposite direction to exiting RNA and TFIIF and its initiation functions continues through the cleft, forming a B‑reader helix and TFIIF was identified in mammalian cells owing to its a B‑linker helix10. A structure of an initially transcribing interaction with Pol II78,79. TFIIF is a heterodimer of Pol II–TFIIB complex containing template DNA and a subunits TFIIFα (also known as RAP74) and TFIIFβ six-nucleotide RNA transcript revealed the entire course (also known as RAP30)80,81. These two subunits cor- of the TFIIB polypeptide in the Pol II cleft30 (FIG. 2a). The respond to Tfg1 and Tfg2 in yeast, respectively82. B‑reader used both its helix and an ordered loop to con- However, yeast TFIIF additionally contains Tfg3, a tact the DNA template strand and was stabilized by the third subunit that is non-essential for transcription83, B‑reader strand that packs against the Pol II lid element. less tightly associated and also a component of other The structural information elucidated how TFIIB69,70 protein complexes84,85. Approximately 50% of Pol II is functions in setting the TSS52,71–75 and revealed new post- associated with TFIIF in a yeast cell86. TFIIF prevents recruitment functions. First, the B‑linker helix is located non-specific interaction of Pol II with DNA87 and sta- in the region above the cleft where DNA opening com- bilizes the PIC88, in particular stabilizing TFIIB within mences10. The B‑linker helix plays a part in DNA open- the PIC76,89. TFIIF also influences TSS selection90, ing and/or the maintenance of the transcription bubble10. stimulates phosphodiester bond formation and early Second, the B‑reader loop binds to the template strand RNA synthesis91–93, and suppresses Pol II pausing94. to position DNA for the initiation of RNA chain synthe- TFIIF further contributes to the stabilization of the sis and to contribute to the recognition of the initiator transcription bubble, and transcription can be initi- DNA sequence30. Third, TFIIB stimulates initial RNA ated to some extent in vitro in the absence of TFIIE synthesis, apparently by allosterically rearranging active and TFIIH but not if TFIIF is also absent95.

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a TFIIB B-core N-terminal B-ribbon cyclin fold C Clamp coiled-coil B-reader B-linker B-linker helix B-reader B-core cyclin folds loop Downstream N C Wall N DNA 1 57 84 123 221 328 345 Non-template N terminus C terminus strand TFIIF Template strand Tfg1 Dimerization domain Charged region N Insertion Insertion WH RNA 1 98 167 305 400 510 673 728 Active N terminus 735 site Tfg2 Dimerization domain Linker WH Side view 1 55 144 192 227 292 354 400

b Yeast core initiation complex Human core initiation complex

Rpb4–Rpb7

Dock Tfg2 WH TFIIB Clamp TFIIFβ WH B-ribbon Non-template strand TBP Downstream B-core DNA C-terminal Template cyclin fold strand Upstream DNA Protrusion TFIIF dimerization domain Top view Top view Figure 2 | Architecture of the core Pol II initiation complex. a | On the left, a ribbon model of the crystal structure of 30 the initially transcribing RNA polymerase II (Pol II)–TFIIB complex (RCSB Protein DataNature Bank Reviews code 4BBS)| Molecular is shown. Cell Biology The structure reveals the different functional domains of TFIIB (green) and their relative locations with respect to Pol II (grey) and nucleic acids. Key elements of TFIIB are labelled, including the B‑linker helix, which is involved in DNA opening and/or open DNA stabilization, and the B‑reader loop, which is assumed to be involved in RNA strand (red) separation from the DNA template (dark blue). The view is from the side25. On the right, the domain architectures of yeast TFIIB and TFIIF are shown. b | Space-filling models of core initiation complexes in yeast12 (left) and humans9 (right) are depicted. The models illustrate how general factors enable Pol II to engage with the promoter. In the yeast complex, the Tfg2 winged helix (WH) domain was also observed near upstream DNA on the outside of Pol II. Comparison of the yeast and human complexes shows that the 3D architecture of the transcription initiation complex is conserved throughout eukaryotes, reflecting the conservation of Pol II and the initiation factors. Pol II is viewed from the top25. TBP, TATA box-binding protein.

The structures of several TFIIF domains have been The TFIIF dimerization module anchors TFIIF to determined. The N‑terminal regions of TFIIFα and Pol II and binds to the Pol II lobe on one side of the TFIIFβ form a dimerization module with a triple barrel cleft near downstream DNA31–33. The WH domain of fold and a protruding β-hairpin, also known as an ‘arm’ Tfg2 is located near the protrusion31,32 and upstream (REF. 96). Both subunits also contain a C‑terminal winged DNA29. The Tfg2 WH domain is mobile in the Pol II– helix (WH) domain97,98. The WH domains are connected TFIIF complex but is located near DNA in initiation to the dimerization module by a charged region in TFIIFα complexes9,12,31. These results were consistent with and a linker region in TFIIFβ31,32,99. In contrast to a canon- early reports that TFIIFβ suffices to recruit Pol II to ical WH domain, the TFIIFα WH domain contains an a promoter complex102,103, and explained why the WH extra α‑helix between helices H2 and H3 (REF. 98), and domain in TFIIFβ is required for function104 and how a pocket composed of H2 and H3 that binds the Pol II it binds to DNA97,105. The Tfg2 WH domain seems to phosphatase FCP1 (also known as CTDP1)100,101. interact with the BRE downstream of the TATA box and with the B‑core region of TFIIB9, which explains Mechanisms of TFIIF function. Structural studies how it stabilizes the upstream promoter complex of Pol II complexes with TFIIF elucidated how and TFIIB76,89. Current data do not support an early TFIIF accomplishes its functions during initiation. EM model of the Pol II–TFIIF complex106.

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a Yeast PIC Human PIC XPD

Rpb4–Rpb7 XPB

TFIIE

TFIIH core Ssl2 TBP

TFIIB Downstream DNA

TFIIA TFIIFβ WH Tfg2 WH TFIIF dimerization domain Top view Top view

b Closed complex Open complex

TBP Fixed upstream Fixed upstream complex complex Point of DNA Transcription melting bubble

TFIIB Ssl2 Ssl2 +1

DNA threaded into cleft +1

Template strand

Active site Side view

Figure 3 | TFIIH and promoter opening. a | Ribbon models of the yeast pre-initiation complex (PIC)8 and the human PIC9 (cryo‑electron microscopy density is shown as a transparent surface) are shown. TheNature Tfg2 Reviews winged helix| Molecular (WH) domain Cell Biology is apparently mobile because it is located near the protrusion in the yeast PIC model8, whereas it is positioned above DNA in the yeast core initiation complex12 (FIG. 2b). The ATPase subunit of TFIIH (known as XPB in humans and Ssl2 in yeast) is located at downstream DNA. Pol II is depicted in grey. b | Models of closed (left) and open (right) RNA polymerase II (Pol II)– TFIIB– TATA box-binding protein (TBP)–DNA complexes based on the Pol II–TFIIB crystal structure (RCSB Protein Data Bank code 3K1F) are shown10. The location of Ssl2 at downstream DNA and the apparent movement of DNA during promoter opening are indicated by arrows. According to the current model for DNA opening, the ATPase Ssl2 functions as a translocase that threads downstream DNA into the active centre cleft of Pol II while upstream DNA remains fixed.

A recent EM study of human initiation complexes9 TFIIE and TFIIH showed that TFIIF stabilizes DNA downstream of the cleft TFIIE and TFIIH are required for promoter DNA open- in a position similar to that proposed by modelling based ing. TFIIH was identified as a factor required for tran- on crystallographic data10 and showed that this occurs scription in vitro111–114. TFIIH contains DNA-dependent along with a minor opening of the Pol II clamp. Both ATPase activity111 that is essential for transcription initia- EM9 and crosslinking12 data indicated that two elements tion2,115. Transcription assays with supercoiled, linear or emanate from the TFIIF dimerization module — the Tfg1 mismatched DNA showed that TFIIH functions in pro- arm and charged helix. The arm and charged helix extend moter opening116 and escape117,118. Additionally, TFIIH into the Pol II cleft near downstream DNA, which sug- functions in the DNA nucleotide excision repair path- gests how they function in TSS selection107,108 and initial way115,119. TFIIE binds to Pol II120 but not to the upstream RNA synthesis91,108,109, respectively. Interactions of TFIIF promoter complex on the TATA box121. TFIIE33 facilitates with DNA both upstream and downstream of the TATA the recruitment of TFIIH to the initiation complex122, box explain early protein–DNA crosslinking patterns34,110. thus providing a bridge between Pol II and TFIIH116,123.

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It also stimulates the DNA-dependent ATPase and kinase associated with the human diseases xeroderma pigmen- activity of TFIIH124. TFIIE alone can open certain pro- tosum, trichothiodystrophy and Cockayne syndrome119. moters and stabilize the open promoter complex116,123, Ssl2 and Rad3 possess helicase activity and exhibit 3ʹ–5ʹ probably by binding to single-stranded DNA125,126. and 5ʹ–3ʹ directionality, respectively147. XPB activity is required for promoter opening in vitro116,148 and in vivo149, TFIIE and its functions. TFIIE is a heterodimer of the whereas the helicase function of XPD is required for DNA subunits TFIIEα and TFIIEβ127,128, which both have opening in the repair pathway150. CDK7 is a kinase that distant homology to the bacterial initiation factor targets the C‑terminal domain of Pol II151–153. The kinase sigma129,130. TFIIEα is composed of an N‑terminal WH module alone is catalytically active but must be located domain, a central zinc-finger domain and a C‑terminal within TFIIH to be specific for the Pol II C‑terminal acidic domain. The N‑terminal half of TFIIEα is suffi- domain154,155. Although C‑terminal domain phosphoryla- cient for interaction with TFIIEβ and transcription function accompanies transcription in vivo, CDK7 activity is tions131. TFIIEβ contains two WH domains that resemble not required for the initiation of transcription in vitro156. a tandem WH domain found in Pol I132 and a basic Structures are available for many TFIIH subunits, C‑terminal region. Structures of the TFIIE domains are including CDK7 (REF. 157) and cyclin H158,159, a domain of available. The structure of the archaeal homologue of the MAT1 (REF. 160), and for archaeal homologues of XPB161 N‑terminal part of TFIIEα, TFE, revealed an extended and XPD162 (see Supplementary information S1 (table)). WH domain that had extra helices on the N and C ter- The overall architecture of TFIIH was revealed by EM mini, and indicated that the domain does not interact analysis142,163,164. Structural information on the position with DNA133. The essential zinc-finger domain from of TFIIH within the PIC suggested mechanisms for how human TFIIEα has a novel topology134. The C‑terminal TFIIH opens promoter DNA (FIG. 3b). Protein–DNA acidic region forms a compact domain135 that is required crosslinking mapped TFIIH in the human PIC in the for strong interaction with TFIIH131, apparently through presence and absence of ATP34. This suggested that TFIIH binding of the pleckstrin homology domain of p62 in acts as a molecular ‘wrench’ that rotates DNA with respect TFIIH135. The first half of the two WH domains of TFIIEβ to a fixed upstream promoter complex at the TATA box, was structurally resolved and shown to bind to DNA thereby creating torque and melting DNA34. As XPB was in vitro136. The C‑terminal region of TFIIEβ also binds to located on DNA downstream of the TATA box and did single-stranded DNA137. not bind to the transcription bubble, TFIIH apparently Structural information on the location of TFIIE on does not function as a conventional helicase, which would Pol II indicates how TFIIE stabilizes the open promoter bind to the unwound region. Mutational analysis of XPB complex. According to crosslinking experiments, TFIIEβ supported the view that DNA opening does not rely on is located just upstream of the TSS138 and TFIIEα also helicase activity165. approaches the DNA in this region139. TFIIE binds to the Pol II clamp domain33, as does archaeal TFE140. Site- Structural models for TFIIH function. Protein specific cleavage analysis showed that the three WH crosslinking in the yeast system indicated that Ssl2 domains of TFIIE are close together within the PIC, with functions as an ATP-dependent, double-stranded DNA the TFIIEα WH domain anchoring the complex to the translocase8. Translocase action on downstream polymerase clamp and the two WH domains in TFIIEβ DNA was suggested to lead to the threading of DNA encircling promoter DNA8. EM of a human PIC con- into the Pol II cleft. The translocase model is consistent taining TFIIE revealed an extended density following a with structural studies of a SWI/SNF-type ATPase166 path from the WH domain of TFIIFβ over DNA and the and with models of closed and open promoter com- clamp to the peripheral Pol II subcomplex RPB4–RPB7 plexes10. It is further consistent with the positioning of (REF. 9). Thus, four WH domains, one from TFIIF and Ssl2 near TFIIE and DNA downstream of the TSS29,167. three from TFIIE, span over the polymerase cleft contain- Trapping of melted DNA in the Pol II cleft may be a key ing loaded DNA9. The TFIIE zinc finger may be located function of TFIIE, because DNA loading into the cleft near the Pol II subcomplex RPB4–RPB7 (REF. 9), which is seems to be reversible168. consistent with data from archaea9,140. A recent EM reconstruction of a TFIIH-containing PIC revealed a distinct density of TFIIH, with two con- TFIIH and promoter DNA opening. TFIIH is a 10‑sub tact points to the remainder of the PIC, one near TFIIE unit factor that, in humans, consists of the ATPase XPB and one near downstream DNA9. This is consistent with a (known as Ssl2 in yeast); a six‑subunit core module com- contact of the TFIIH core to TFIIE169. Compared with the prising XPD (also an ATPase; Rad3 in yeast), p62 (Tfb1 in position of Ssl2 derived in the yeast crosslinking study8, yeast), p52 (Tfb2 in yeast), p34 (Tfb4 in yeast), p8 (Tfb5 XPB was located approximately 10 bp further down- in yeast) and p44 (Ssl1 in yeast); and the three‑subunit stream, and it was suggested that XPB ‘walks’ on the DNA kinase module CDK7–cyclin H–MAT1 (Kin28–Ccl1– away from the PIC, causing straining that facilitates Tfb3 in yeast) (see TABLE 1 for more details)141,142. Only the DNA melting. Although the EM work did not reveal the complete TFIIH is transcription-competent, whereas TFIIH kinase module, comparison with a reconstruction the kinase module is not required for DNA repair143,144. of TFIIH alone indicated that it would be located close to Three TFIIH subunits have catalytic activity. XPB and the C‑terminal domain linker and TFIIE9, which is con- XPD and their yeast homologues are ATPases145,146. sistent with the stimulation of C‑terminal domain phos- Mutations in the genes encoding XPB and XPD are phorylation by TFIIE124. An alternative TFIIH location

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a Yeast b –120 to –40 +1 TBP

TATA box Inr TAND2 TBP TAF1–TAF2 TAF6–TAF9 TFIIA Drosophila and humans –30 +1 +30 DCE BREu BREd TATA DNA

TATA box Inr MTE DPE TAND1 c Lobe B TAF4 d N terminus

TAF5 WD40 repeats TAF9 Downstream DNA

TAF6 90°

Lobe C TAF4 TAF12 Lobe A TAF5 Upstream DNA TAF5 N terminus Figure 4 | TFIID and promoter specificity. a | Some DNA elements in core promoters from yeast (top) and Drosophila Nature Reviews | Molecular Cell Biology and humans (bottom) are depicted, and elements known to contact TFIID are shown. b | Superimposition of DNA complex structures shows that the TAF1 (a TATA box-binding protein (TBP)-associated factor) regions TAND1 (TAF1 N-terminal domain 1) and TAND2 would clash with TATA DNA and TFIIA, respectively195–198. Thus, binding of TBP to this region of TAF1 and DNA–TFIIA is mutually exclusive. Modelling is based on structures with RCSB Protein Data Bank codes 4B0A (TBP–TAF1 complex) and 1NH2 (TFIIA–TBP–DNA complex). c | Low-resolution electron microscopy‑derived architecture of endogenous human TFIID bound to a large promoter DNA208 with a TATA box (red), Initiator (Inr, which is a sequence containing the transcription start site; purple) sequence, motif ten element (MTE; dark green) and downstream promoter element (DPE; light green) is shown. d | An electron microscopy structure of a recombinant human TFIID core complex, revealing the location of several TAF subunits, is shown211. The core complex shows twofold symmetry, but association with additional TAF subunits breaks this symmetry (not shown). The relative position of TFIID within the pre-initiation complex

remains to be elucidated. BREd, downstream B recognition element; BREu, upstream B recognition element; DCE, downstream core element.

derived for the yeast PIC is inconsistent with that derived TFIID and promoter recognition. TFIID is involved by the above studies and instead suggests that TFIIH is in the recognition of core promoter sequences (FIG. 4a). positioned above the Pol II cleft13. It was originally thought that TFIID was recruited to Pol II promoters through the TATA-binding activity of TFIID and promoter specificity TBP. A conserved TATA box is, however, found only TFIID is a conserved, multifunctional general tran- in 10–20% of yeast and human promoters180. Analysis scription factor involved in promoter recognition, of core promoter sequences led to the identification of PIC assembly and chromatin remodelling (reviewed several additional core promoter elements that are rec- in REFS 170,171). TFIID is composed of TBP and ognized by TAFs (reviewed in REFS 181,182). The initia- 13–14 TAFs, with a total size of approximately 1.2 MDa. tor element, which is found in yeast and metazoans180,183, In addition to TBP and the 13 canonical TAFs that are overlaps with the TSS and can be recognized by TAF1– conserved from yeast to humans172, metazoan genomes TAF2 (REF. 184). The motif ten element (MTE)185 and the encode several cell type-specific variants of TBP and TAFs downstream promoter element (DPE)186 were discovered that form alternative TFIID complexes that are required in Drosophila melanogaster and later also identified in for the expression of subsets of genes173–176. Experiments in human promoters63. Both elements are probably rec- yeast have indicated that TFIID contributes to the expres- ognized by TAF6–TAF9 (REFS 187,188). An additional sion of most Pol II‑transcribed genes177. At metazoan pro- overlapping promoter element, the downstream core moters, diversity in TFIID complex composition depends element (DCE), was identified in human promoters189 on the developmental stage and cell type178,179. and may be contacted by TAF1 (REF. 190). TFIID also

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binds to activating transcription factors191,192, which and EM reconstructions of TFIID have generally been at help to recruit TFIID to promoters and may facilitate resolutions that are too low for crystal structures to be fit enhancer–promoter interactions. unambiguously. A step towards overcoming this prob- TFIID is also able to modulate TBP activity. The lem was taken in a recent study in which a core com- N‑terminal domain of TAF1 binds to TBP and inhib- plex of human TFIID, comprising approximately 50% its its binding to TATA DNA193. The TAF1 N terminus of the mass of TFIID, was expressed recombinantly211. contains two conserved subdomains, TAF1 N-terminal A more rigid TFIID core with two-fold symmetry could domain 1 (TAND1) and TAND2, which interact with be resolved at 12 Å resolution, and most densities were TBP194. TAND1 binds to the DNA-binding surface of explained using known structures (FIG. 4d). A highly TBP in a way that mimics TATA DNA, whereas TAND2 intertwined architecture containing many protrusions binds to the convex surface of TBP competitively with and a large surface area was revealed. In the core TFIID TFIIA195–198 (FIG. 4b). Binding of TFIIA displaces the structure, histone folds enabled TAF–TAF interactions inhibitory TAF1 domain and stabilizes the TBP–pro- but histone octamer geometry was not observed. The moter interaction199. In this way, TFIID is able to fine- symmetry of the core TFIID complex was broken upon tune DNA binding to promoters containing different addition of TAF8 and TAF10, which would enable the combinations of binding elements. remaining TAFs to assemble along the periphery.

Structural studies of TFIID. Crystallographic studies Conclusions and perspectives of TAF subunits and EM studies of large TFIID com- The initiation of transcription at Pol II promoters is a plexes have been conducted. Early analysis of TAF very complex process in which dozens of polypeptides subunits revealed the presence of several histone fold cooperate to recognize and open promoter DNA, locate domains200,201. This, in conjunction with biochemi- the TSS and initiate pre-mRNA synthesis. Because of its cal and crystallographic studies, led to the proposal of large size and transient nature, the study of the Pol II ini- a histone octamer-like core for TFIID202–204. Early EM tiation complex will continue to be a challenge for struc- studies of complete human and yeast TFIID complexes tural biologists. The first decade of work, which started revealed a conserved three-lobed, horseshoe-like struc- in the 1990s, provided structures for many of the factors ture of TFIID205–207. Studies of endogenous TFIID with involved and several of their DNA complexes. The sec- additional factors provided the initial information on ond decade of research provided structural information the overall organization of promoter-bound TFIID on Pol II complexes and led to models for how general (FIG. 4c). TFIID purified from Saccharomyces pombe, transcription factors function. Over the next decade, we Saccharomyces cerevisiae and Homo sapiens has been hope that a combination of structural biology methods investigated in complexes with promoter DNA191,208,209. will resolve many remaining questions on transcription In an investigation of yeast TFIID in a complex initiation, and elucidate the mechanism of promoter with TFIIA, DNA and the activator Rap1, activator- opening and initial RNA synthesis, the remodelling dependent DNA looping was observed191. In a study of of the transient protein–DNA interactions occurring human TFIID in a complex with TFIIA and a ration- at various stages of initiation, and the conformational ally designed DNA construct that combined various changes underlying the allosteric activation of initia- promoter elements210, the largest continuous promoter tion and the transition from initiation to elongation. DNA density bound to TFIID was obtained208. Important next steps include more detailed structural Low amounts of an intact complex coupled with the characterizations of TFIIH and the 25‑subunit coactiva- large size and flexibility of endogenously purified TFIID tor complex Mediator, not only in their free forms but have made structural studies of holo-TFIID challenging, also as parts of initiation complexes.

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