Transcription of eukaryotic protein-coding genes begins with assembly of the RNA polymerase (Pol) II initiation complex and promoter DNA opening. Here we report cryo-electron microscopy (cryo-EM) structures of yeast initiation complexes containing closed and open DNA at resolutions of 8.8 Å and 3.6 Å, respectively. DNA is positioned and retained over the Pol II cleft by a network of interactions between the TATA-box-binding protein TBP and transcription factors TFIIA, TFIIB, TFIIE, and TFIIF. DNA opening occurs around the tip of the Pol II clamp and the TFIIE ‘extended winged helix’ domain, and can occur in the absence of TFIIH. Loading of the DNA template strand into the active centre may be facilitated by movements of obstructing protein elements triggered by allosteric binding of the TFIIE ‘E-ribbon’ domain. The results suggest a unified model for transcription initiation with a key event, the trapping of open promoter DNA by extended protein–protein and protein–DNA contacts.
At a glance
For transcription initiation, Pol II assembles with the basal transcription factors (TF) IIB, TFIID (or its subunit TBP), TFIIE, TFIIF, and TFIIH1, 2, 3, 4 on double-stranded promoter DNA to form the closed complex (CC). Upon DNA opening, the template strand slips into the Pol II active centre, and a DNA ‘bubble’ forms ~20–30 base pairs (bp) downstream of the TATA box5, leading to the open complex (OC). Efficient DNA opening requires TFIIE and TFIIH6, 7, 8, but these factors are not required for low levels of transcription9, 10 and TFIIE alone can open certain promoters11, 12. Subsequent RNA synthesis results in the initially transcribing complex (ITC), which is then converted into an elongation complex for processive RNA synthesis.
The three-dimensional architecture of Pol II initiation complex intermediates was studied in yeast13, 14, 15, 16, 17, 18, 19 and human20 systems. These studies revealed that the promoter assembly containing TBP and TFIIB resides over the Pol II wall and positions DNA above the polymerase cleft and along the clamp. TFIIE and TFIIF bind to opposite sides of Pol II and flank promoter DNA. Owing to the limited resolution of available structural studies, many questions regarding the molecular basis of initiation remain, including the mechanisms of DNA opening and template-strand loading into the active centre. A differing model of the yeast initiation complex17 was revised recently21, and now agrees, on a topological level, with other studies and results reported here.
Here we report cryo-EM structures of CC and OC assemblies from the yeast Saccharomyces cerevisiae at resolutions of 8.8 Å and 3.6 Å, respectively. The structures contain all of the basal transcription factors except TFIIH, which is currently not available at a quality required for high-resolution analysis. We show that DNA opening can occur in the absence of TFIIH, and provide mechanistic insights into DNA opening and template-strand loading. Our results also unveil the exact location and intricate structure of basal factors and their induced folding interactions with each other, with Pol II, and with promoter DNA. The data also demonstrate the high structural conservation between yeast and human initiation systems.
Transcription initiation complex at 3.6 Å
We extended our previous cryo-EM analysis of a Pol II core initiation complex containing TBP, TFIIB, and TFIIF19 by adding TFIIA and TFIIE (Methods). Formation of a stable and stoichiometric complex required the presence of core Mediator, which showed low occupancy and high flexibility under cryo-EM conditions, as observed previously19, and was excluded from further analysis (Methods, Extended Data Fig. 1a–c). We acquired 257,259 cryo-EM single particle images using a K2 direct electron detector (Methods, Extended Data Fig. 1b, c). Unsupervised particle sorting led to an OC structure at an overall resolution of 3.6 Å, revealing the Pol II core at up to 3.1 Å (OC1), compared to 7.8 Å for our core ITC structure19 (Fig. 1a, b, Extended Data Figs 1d–g). Further particle sorting revealed improved density for TFIIB and TFIIF at around 4 Å resolution (OC2 and OC4), and for TFIIE at 4.4 Å resolution (OC3, Methods, Extended Data Figs 1i, k, l). The final structure mainly consists of atomic models (90%) and contains backbone models for parts of the basal factors (Extended Data Fig. 1f–l).
The structure reveals upstream DNA above the Pol II wall and downstream DNA in the active centre cleft19. We observed only fragmented density for single strands within the DNA bubble (Fig. 1b, c, Extended Data Fig. 1m), and no density for RNA, which was not retained during cryo-EM sample preparation (not shown). The bubble contains 15 mismatches, begins at the natural distance of ~20 bp downstream of the TATA box5, and extends further downstream than when it initially forms, resembling the situation during transcription start-site scanning. For the basal factors, regions that are essential for cell viability are generally observed, whereas non-essential, non-conserved regions16, 22, 23, 24, 25, 26 are often mobile (Fig. 1a, b, Extended Data Fig. 1 j–l). TFIIA is located near upstream DNA and TBP as expected23 (Fig. 1b, Extended Data Fig. 1j). For TFIIB, the B-ribbon and B-core domains are well defined, including the newly modelled carboxy-terminal cyclin domain from yeast (Fig. 2), whereas the B-linker shows weak density, and the B-reader15, 19 is mobile (Extended Data Fig. 1i).
DNA positioning and retention
TFIIF adopts an intricate fold within the OC. Its dimerization module and charged helix are located on the Pol II lobe domain as in the ITC18, 19 (Extended Data Fig. 2). The ‘arm’ in the large TFIIF subunit Tfg1 (human RAP74) adds a β-strand to the Pol II protrusion, and projects into the cleft, where it may stabilize the DNA bubble18, 19, 20 (Fig. 2). The linker in TFIIF subunit Tfg2 (human RAP30) emanates from the dimerization module, and winds along the base of the protrusion, where it binds a hydrophobic pocket. The Tfg2 linker continues between the protrusion and the TFIIB cyclin domains, and connects to the Tfg2 C-terminal winged-helix (WH) domain on top of the cleft (Fig. 2, Extended Data Fig. 2f). The Tfg2 linker stabilizes TFIIB on the wall of Pol II27, 28. The yeast-specific amino-terminal region of Tfg1 binds near the Pol II external 1 domain, according to a separate crystallographic analysis (Extended Data Fig. 2d, e, Extended Data Fig. 9c).
TFIIE is located between the clamp and the Rpb4–Rpb7 stalk (Fig. 3a, b, Methods, and Extended Data Fig. 3), consistent with previous topological placement of TFIIE16, 20, 29, 30, 31 and its archaeal counterpart32. The TFIIE structure differs from a recent model obtained at 6 Å resolution21 (see Extended Data Fig. 1e legend for details, Extended Data Fig. 3, and refs 16, 17). The large TFIIE subunit Tfa1 (human TFIIEα) contains an extended winged helix (‘eWH’) domain33 and a zinc ribbon domain34 (‘E-ribbon’) that are connected by α-helices (called here ‘E-linker’) (Fig. 3b). The eWH domain uses its helix α3 to contact both the tip of the Pol II clamp helices and the DNA backbone at positions −13/−14 upstream of the transcription start site (TSS, position +1) (Fig. 1c, 3a). The E-ribbon binds between the clamp, the Rpb7 oligonucleotide-binding (OB) domain, and the B-ribbon (Fig. 3a). The E-linker20, 21 and the mobile C-terminal domain of Tfa1 contact TFIIH7, 35, which may alter TFIIE conformation. The small TFIIE subunit Tfa2 (human TFIIEβ) contains two WH domains (‘WH1’ (ref. 36), ‘WH2’), and two conserved α-helices (called here ‘E-tether’) that bind the E-linker (Fig. 3b). Consistent with the structure, the E-tether is essential for TFIIE function16, 37 and subunit dimerization (Extended Data Fig. 3a, refs 16, 38). The structure further indicates that TFIIE must be displaced or at least moved before the elongation factor Spt4/5 (human DSIF) can bind to polymerase32, 39.
The OC structure thus reveals how TFIIF and TFIIE bind open promoter DNA from opposite sides of the Pol II cleft. First, TFIIF adopts an extended induced structure that allows it to retain the upstream DNA–TBP–TFIIB assembly on the wall and to bind the DNA bubble and downstream duplex in the cleft (Figs 1b, 2). Second, the TFIIF Tfg2 WH domain and the TFIIE Tfa2 WH1 domain contact each other above upstream DNA to encircle and retain DNA. Third, the eWH domain of TFIIE binds DNA in the region of initial DNA opening and its long β1–β2 hairpin33 (called here the ‘E-wing’) projects to the upstream edge of the bubble (Fig. 3a), suggesting that the eWH domain stabilizes open DNA. Taken together, the highly modular and flexible basal factors TFIIF and TFIIE undergo substantial induced folding transitions to engage in multiple protein–DNA and protein–protein interactions to stabilize the OC.
DNA opening and loading
Modelling of a closed DNA promoter onto the OC structure shows that closed DNA would clash with the TFIIE eWH domain. This suggests that the eWH domain adopts a different position before DNA opening. To investigate this, and to provide insights into the transition from the CC to the OC, we repeated structure determination with closed DNA instead of pre-opened DNA (Methods, Extended Data Fig. 4). Surprisingly, cryo-EM analysis revealed that about 3 out of 4 particles contained open DNA, although closed DNA was used for complex preparation. DNA opening occurred in the absence of TFIIH (Extended Data Fig. 4h, i). From these particles we obtained an independent reconstruction of the spontaneously formed OC at 6.1 Å resolution (OC5, Extended Data Fig. 4h). Weak density for upstream and downstream DNA segments indicates that DNA bubbles of various sizes formed during DNA opening (Extended Data Fig. 4i). The reconstruction resembles the high-resolution OC structure, suggesting that the latter was not perturbed by the use of pre-opened DNA (Extended Data Fig. 4h).
From the remaining particle images we obtained a reconstruction of the CC at 8.8 Å resolution (Fig. 4a). Comparison of this CC reconstruction with the OC structure reveals movements mainly in TFIIE (Fig. 4b, Extended Data Fig. 4e–g). In the CC, the E-wing lies on top of the DNA around position –7, in the region where DNA opening begins7 (Extended Data Fig. 4e). Consistent with this contact, Tfa1 (or the human counterpart TFIIEα) crosslinks to DNA near this point in the CC30, 40, 41 (Extended Data Fig. 4e). Conversion of the CC to the OC involves movement of upstream DNA and the DNA-associated domains Tfa2 WH1 and Tfg2 WH towards the cleft. DNA opening allows the TFIIE eWH domain to bind the tip of the clamp, and enables the E-wing to move near the upstream edge of the DNA bubble. Consistent with these changes, crosslinks between upstream DNA and the large TFIIE subunit are altered when the CC is converted to the OC40. These observations indicate that DNA opening involves TFIIE, and in particular the eWH domain.
TFIIE may also be involved in loading of the DNA template strand into the active centre during the transition from the CC to the OC. In previous structures, the path for loading the template was obstructed by the TFIIB B-reader and the Pol II fork loop 1 and lid. However, the B-reader is mobile in the OC structure, and fork loop 1 and the lid are moved to provide a path for template-strand loading (Fig. 5b, Extended Data Fig. 5a–c). These movements are apparently triggered by TFIIE because binding of the E-ribbon leads to a shift in the B-ribbon that partially withdraws the B-reader from the cleft (Fig. 5c). Thus allosteric binding of the E-ribbon apparently induces ‘clearance’ of the Pol II cleft that may facilitate template-strand loading into the active centre, and transcription start-site scanning in yeast.
To support the proposed functions of structural elements in TFIIE, we prepared recombinant TFIIE variants and tested them for binding to the CC and for promoter-dependent transcription activity in yeast nuclear extract (Methods, Fig. 3c, Extended Data Fig. 3g–i). Mutation of only three surface residues in the E-ribbon that contact the Pol II subunit Rpb7 strongly impaired both binding to the CC and transcription activity, and led to a severe growth defect in yeast (Fig. 3c, Extended Data Fig. 3h, j). Further, the eWH domain is required for TFIIE function16, and disruption of the eWH contact with DNA by introducing negatively charged glutamate residues in the eWH helix α3 leads to a transcription defect and impairs binding to the CC (Fig. 3c, Extended Data Fig. 3h). Other mutations in the eWH domain did not show functional defects in our assays. Deletion of the E-wing results in a mild growth phenotype (Extended Data Fig. 3j), and does not impair in vitro transcription (Fig. 3c), maybe because TFIIH compensates for the loss of E-wing function in these assays.
Model of transcription initiation
Structural comparisons of the yeast CC, OC, and ITC19 with each other and with the highly conserved human CC20 reveal differences in the positions of DNA, the clamp, and TFIIE, and lead to an extended model of DNA opening (Fig. 6a, b, Extended Data Fig. 6). In this model, promoter DNA is initially bent away from the active site by ~20° near position −10 at the tip of the clamp helices (yeast CC). The clamp then opens slightly (Extended Data Fig. 6b), allowing promoter DNA to bend in the opposite direction and to enter the upper part of the cleft (‘human CC’). This frees the site at the tip of the clamp helices that can now bind the TFIIE eWH domain. DNA can then no longer swing back to its initial position, because this would result in a steric clash with the repositioned eWH domain. However, when DNA opening occurs around position −10, the upstream DNA can swing back to its original position and this stabilizes the DNA duplex single-strand junction. TFIIH then rotates downstream DNA and pushes the template single strand into the cleft using its ATP-dependent translocase activity, as suggested previously16. When the bubble extends downstream5, the template strand is loaded into the active centre cleft via a path cleared by allosteric binding of TFIIE (yeast OC). Template-strand loading allows the clamp to close again and to trap downstream DNA in the cleft (Extended Data Fig. 6b). The B-reader then covers the template strand, and helps to detect the transcription start site13, triggering RNA synthesis (yeast ITC).
This model explains how DNA opening can be achieved with the use of binding energy alone, at least at some promoters11, 12. DNA opening allows for new protein interactions at the upstream DNA duplex single-strand junction involving the eWH domain, the TFIIF arm, and the Pol II clamp. DNA loading into the cleft also enables new interactions of the downstream DNA duplex with the TFIIF charged helix and the Pol II cleft and clamp. These additional contacts may compensate for the energy needed for DNA melting and help to trap open DNA and to prevent its re-closure.
A similar mechanism for ATP-independent DNA opening may be used in other transcription systems. Proteins with homologies to TFIIB, TFIIE, and TFIIF, but not TFIIH, are present in the Pol I and Pol III systems42, and counterparts of TBP, TFIIB, and TFIIE are found in archaea38. The bacterial transcription initiation system is structurally unrelated, but conceptually similar. DNA opening occurs spontaneously above the cleft, the open DNA is trapped with the use of binding energy, and this requires clamp opening and closure43, 44, 45, 46, 47. DNA opening at a subset of bacterial genes however requires the ATP-dependent initiation factor σ54, and this enables further regulation48. Similarly, Pol II regulation can occur at the level of DNA opening49, and TFIIH is required to keep DNA open at least at selected promoters8. This suggests that the Pol II initiation system evolved to depend on the ATP-consuming factor TFIIH for DNA opening, probably in response to an expanded need for gene regulation.
No statistical methods were used to predetermine sample size. The experiments were not randomized and the investigators were not blinded to allocation during experiments and outcome assessment.
Vectors and sequences
The open reading frame (ORF) encoding full-length TBP was amplified from Saccharomyces cerevisiae (Sc) genomic DNA and cloned into a pOPINE vector, containing a C-terminal 6×histidine tag. Codon-optimized Tfg1, Toa1, and Toa2 were commercially obtained for expression in Escherichia coli (E. coli, Life Technologies). Toa1 (Δ95–209) was obtained by quick-change PCR and cloned with Toa2 into a pOPINE vector, containing a C-terminal 6×histidine-tag on Toa2. Tfa1 and Tfa2 ORFs were amplified from genomic Sc DNA and cloned sequentially into a pET vector. ORFs for Tfg1 and Tfg2 were cloned sequentially into a modified pET-Duet-1 vector, containing an N-terminal 10×histidine-8×arginine-SUMO-tag on Tfg1. Additional ribosomal binding sites were introduced as described51. For heterologous co-expression of Sc 16-subunit core Mediator (cMed)-Med1, 13-subunit cMed lacking Med4–Med9 was cloned with previously described vectors19. Three-subunit Med1–Med4–Med9 was prepared by cloning Sc ORFs of Med1, Med4, and Med9 into a modified pET-Duet-1 vector, containing an additional N-terminal 10×histidine-8×arginine-SUMO-tag on Med1. Sequences are available upon request.
All proteins were expressed in E. coli BL21(DE3)RIL cells (Stratagene). The identity of all purified proteins was confirmed by mass spectrometry. All purified proteins and complexes were flash-frozen and stored at −80 °C.
Full-length TBP was expressed in E. coli that were grown in lysogeny broth (LB) medium at 37 °C to an optical density (OD) of 0.5 at 600 nm, and induced with 0.5 mM isopropyl-β-D-thiogalactoside (IPTG) for 4 h at 20 °C. Cells were lysed by sonication in buffer A (25 mM HEPES (pH 7.5), 500 mM KCl, 10% glycerol, 2.5 mM dithiothreitol (DTT)) containing protease inhibitors52. The soluble fraction was applied to a 5 ml Histrap HP column, washed with buffer A containing 1 M KCl, and eluted with buffer A containing 350 mM imidazole. The sample was diluted 1:4 with buffer A lacking KCl and applied to anion-exchange chromatography using a MonoS 5/50 (GE Healthcare), and eluted with a linear gradient of buffer A from 100–1000 mM KCl. TBP was further purified by size-exclusion chromatography using a Superose 12 10/300 (GE Healthcare) column, equilibrated in buffer B (10 mM HEPES (pH 7), 200 mM NaCl, 5% glycerol, 2 mM DTT). TBP-containing fractions were pooled and concentrated to 8 mg ml−1.
TFIIA was obtained by co-expression of its subunits Toa1 and Toa2 in E. coli. Transformed cells were grown in LB medium at 37 °C to an OD of 0.5 at 600 nm. Expression was induced with 0.5 mM IPTG at 37 °C for 4 h. Cells were lysed in buffer C (25 mM Tris-HCl (pH 8.0), 500 mM NaCl, 10% glycerol, 2 mM DTT) containing protease inhibitors52, and cleared by centrifugation. The supernatant was applied to a 5-ml HisTrap HP column, washed with buffer C containing 1 M NaCl, and eluted with buffer C containing 250 mM imidazole. Fractions containing the complex were pooled, diluted 1:4 with buffer C lacking NaCl, loaded on a MonoQ 5/50 anion-exchange column, and eluted with a linear gradient of buffer C from 100–500 mM NaCl. TFIIA containing fractions were pooled and applied to a Superdex 75 10/300 column (GE Healthcare), in buffer B. TFIIA was concentrated to 8 mg ml−1. TFIIB was prepared as described15.
Recombinant TFIIE was obtained by co-expression of its subunits Tfa1 and Tfa2 in E. coli. Cells were transformed and grown in LB medium at 37 °C to an OD of 0.6 at 600 nm, and expression was induced by 0.5 mM IPTG for 18 h at 18 °C. Cells were lysed by sonication in buffer D (50 mM Tris-HCl (pH 8.0), 300 mM NaCl, 0.02% Tween-20, 5 mM DTT) containing protease inhibitors52. The lysate was cleared by centrifugation and applied to a 5 ml Histrap HP column, equilibrated in buffer E (buffer M, lacking Tween-20, containing 10 mM imidazole). The column was washed with 10 column volumes of buffer E and eluted with buffer E containing 250 mM imidazole. TFIIE was then subjected to anion-exchange chromatography using a 5-ml Hi-Trap Heparin column (GE Healthcare), equilibrated in buffer F (50 mM Tris-HCl (pH 8.0), 100 mM NaCl, 2% glycerol, 5 mM DTT). The complex was eluted with a linear gradient of buffer F from 100–2000 mM NaCl. To improve purity, TFIIE was further applied to a Superose 12 10/300 size-exclusion column, in buffer G (5 mM HEPES (pH 7.25), 40 mM ammonium sulphate, 10 μM ZnCl2, 10 mM DTT). TFIIE containing fractions were pooled and concentrated to 9.6 mg ml−1.
Sc TFIIF subunits Tfg1 and Tfg2 were co-expressed in E. coli and cells were grown in LB medium at 37 °C to an OD of 0.8 at 600 nm. Expression was induced with 0.2 mM IPTG for 3 h at 37 °C. Cells were lysed by sonication in buffer H (50 mM HEPES (pH 7.0), 350 mM KCl, 10% glycerol, 2 mM DTT) supplemented with 50 mM imidazole and protease inhibitors52. Cleared lysate was applied to a 5-ml HisTrap HP column equilibrated in buffer H. The column was washed with 8 column volumes of buffer H containing 1000 mM KCl, and eluted with a linear gradient from buffer H to buffer I (50 mM HEPES (pH 7.0), 250 mM KCl, 800 mM imidazole, 10% glycerol, 2 mM DTT). The conductivity of the eluate was adjusted to match that of buffer J (50 mM HEPES (pH 7.0), 150 mM KCl, 10% glycerol, 2 mM DTT) and 3C protease cleavage was carried out for 2 h. The complex was then applied to cation-exchange chromatography using a 1-ml HiTrap SP HP column (GE Healthcare), equilibrated in buffer J, and eluted in a linear gradient from 150–1000 mM KCl. TFIIF was further purified by size-exclusion chromatography using a Superdex 200 10/300 Increase column (GE Healthcare), in buffer K (10 mM MES (pH 6.2), 150 mM KCl, 10% glycerol, 2 mM DTT). Purified TFIIF was concentrated to 3.9 mg ml−1. For previous studies of the initiation complex we used the conserved S. mikatae Tfg1 (refs 18, 19), owing to difficulties in recombinant expression of its Sc homologue. Here, expression of Sc Tfg1 was enabled by codon optimization.
For preparation of recombinant 16-subunit cMed–Med1, 13-subunit cMed lacking Med4–Med9 was prepared essentially as described19. Separately, a plasmid containing Med1–Med4–Med9 was transformed in E. coli and cells were grown in LB medium at 37 °C to an OD of 0.6 at 600 nm. Protein expression was induced with 0.5 mM IPTG at 18 °C for 24 h. Cells were collected and lysed by sonication in buffer L (25 mM HEPES (pH 7.5), 400 mM potassium acetate, 10% glycerol, 20 mM imidazole (pH 8), 2 mM DTT) containing protease inhibitors52. Lysate was cleared by centrifugation and applied to a 5-ml Histrap HP column (GE Healthcare), equilibrated in buffer M (25 mM HEPES (pH 7.5), 400 mM potassium acetate, 10% glycerol, 30 mM imidazole (pH 8), 2 mM DTT). The column was washed with 5 column volumes of buffer M and a linear gradient over 10 column volumes from 30–100 mM imidazole. The heterotrimer was eluted with 300 mM imidazole over 10 column volumes. Fractions containing the complex were diluted 1:3 with buffer N (25 mM HEPES (pH 7.5), 100 mM KCl, 10% glycerol, 1 mM EDTA, 5 mM DTT) and incubated with 3C protease for 2 h on ice, to remove the affinity tag. The complex was further purified by anion-exchange chromatography using a MonoQ 5/50 GL column (GE Healthcare), equilibrated in buffer N, and was eluted with a linear gradient from 100–600 mM KCl over 150 column volumes. Fractions containing the complex were pooled and applied to size-exclusion chromatography using a Superose 6 10/600 column (GE Healthcare), equilibrated in buffer O (25 mM HEPES (pH 7.5), 200 mM KCl, 5 mM DTT). Purified Med1–Med4–Med9 complex was concentrated to 2.7 mg ml–1. To prepare 16-subunit cMed–Med1, 13-subunit cMed was incubated with a twofold molar excess of Med1–Med4–Med9 for 30 min at 25 °C and 30 min on ice, and purified by size-exclusion using a Superose 6 10/600 column, in buffer P (25 mM HEPES (pH 7.5), 400 mM KCl, 5% glycerol, 5 mM DTT). Fractions containing cMed–Med1 were pooled and concentrated to 2 mg ml−1.
Preparation of initiation complexes
Yeast 12-subunit Pol II was prepared as described53. Open and closed initiation complexes were prepared with several modifications of the previous cITC–cMed assembly scheme19 and with a different nucleic acid scaffold for the closed complex. The 72 nucleotide nucleic acid scaffold previously used to prepare the Pol II core ITC (cITC)19 contains a 15 nucleotide mismatch transcription bubble and six nucleotide RNA, and was used for assembly of the open initiation complex (OC). The closed complex (CC) contained a nucleic acid scaffold with 13 nucleotide longer downstream DNA, based on the HIS4 promoter (template, 5′-TGATATTTTTATGTATGTACAACACACATCGGAGGTGAATCGAACGTTCCATAGCTATTATATACACAGCGTGCTACTGTTCTCG-3′; non-template, 5′-CGAGAACAGTAGCACGCTGTGTATATAATAGCTATGGAACGTTCGATTCACCTCCGATGTGTGTTGTACATACATAAAAATATCA-3′). The initiation complex was prepared as follows. Pol II (200 μg at 3 mg ml−1) was incubated with a fourfold molar excess of TFIIF. A twofold molar excess of nucleic acid scaffold over Pol II, tenfold molar excess of TFIIA, fourfold molar excess of TBP and TFIIB were added to buffer Q (25 mM HEPES-KOH (pH 7.5), 150 mM potassium acetate, 5% glycerol, 2 mM MgCl2, 5 mM DTT) and incubated with pre-formed Pol II–TFIIF complex for 8 min at 25 °C. TFIIE was added in a tenfold molar excess over Pol II and incubated for 5 min at 25 °C. cMed–Med1 was added in a 1.2-fold molar excess over Pol II and incubated for 50 min at 25 °C. Open and closed complexes were purified using a Superose 6 3.2/300 size exclusion column (GE Healthcare), equilibrated in buffer Q. Fractions containing the complex were pooled (0.4–0.8 mg ml−1) and additionally incubated with equimolar amounts of nucleic acid scaffold. The sample was then crosslinked for 30 min on ice using 0.1% glutaraldehyde (Electron Microscopy Sciences), and the reaction was quenched with 50 mM lysine (Sigma). The crosslinked sample was re-purified in a second size-exclusion step using a Superose 6 3.2/300 column, equilibrated in buffer Q lacking glycerol. Fractions containing initiation complexes were pooled (0.2–0.6 mg ml−1) and used for EM grid preparation.
Initiation complex samples were applied to R3.5/1 holey carbon grids (Quantifoil). Grids were glow-discharged for 15 s before deposition of 4.5 μl complex, and subsequently blotted and vitrified by plunging into liquid ethane with a Vitrobot Mark IV (FEI) operated at 4 °C and 100% humidity. Cryo-EM data was acquired on a FEI Titan Krios operated in EFTEM mode at 300 keV, and equipped with a K2 Summit direct detector (Gatan). Automated data collection was carried out using the TOM toolbox54 to acquire 1756 movies of the OC with a range of defocus values (from −0.7 μm to −4.2 μm) at a nominal magnification of 37,000× (1.35 Å per pixel). The camera was operated in ‘super-resolution’ mode (0.675 Å per pixel), with a total exposure time of 10 s fractionated into 25 frames, a dose rate of ~5 e− per pixel per second, and total dose of 33 e− Å−2 per movie. Cryo-EM data of the CC was collected in the same manner, except that 959 movies were acquired with a defocus range from −0.8 μm to −5 μm, a total exposure time of 6 s with 20 frames, a dose rate of ~8 e− per pixel per second, and total dose of 40 e− Å−2. Movies were aligned as described19, 55, except that images were not partitioned into quadrants.
For single particle analysis of the OC, an initial set of 10,225 particles was selected semi-automatically using e2boxer.py from EMAN2 (ref. 56). CTF parameters were estimated using CTFFIND4 (ref. 57). CTF correction and subsequent image processing was performed with RELION 1.3 (ref. 58), unless otherwise noted. Resolution was reported on the basis of the gold-standard Fourier shell correlation (FSC) (0.143 criterion) as described previously59 and temperature factors were determined and applied automatically in RELION58. Selected particles were extracted with a 3002 pixel box and pre-processed to normalize images. Reference-free two-dimensional (2D) class averages were calculated, and twelve representative classes were low-pass filtered to 25 Å resolution and used as templates for automated picking60 of all micrographs. The resulting 415,030 particle images were screened manually and by reference-free 2D classification, yielding 257,259 particle images that were used for subsequent processing. The 7.8 Å cryo-EM map of the yeast cITC19 (EMD-2785) was low-pass filtered to 50 Å and used as initial model for 3D refinement of the 10,225 particle set. This revealed an OC density to an estimated resolution of 10 Å. This density was low-pass filtered to 50 Å and used for processing of the complete OC cryo-EM data set (Extended Data Fig. 7a–d). A 3D reconstruction of all particles was calculated to 3.75 Å, and subjected to particle polishing using RELION 1.4 beta58. This lead to an improved density at 3.58 Å resolution.
Hierarchical 3D classification was carried out without image alignment, to reduce computational requirements and identify homogeneous single particle groups (Extended Data Fig. 7a–d). Soft masks encompassing the complete OC or smaller regions of Pol II and basal factors were generated using the volume eraser in UCSF Chimera61 and RELION58. This included masks for Pol II stalk–TFIIE E-ribbon (Extended Data Fig. 7a, c), upstream DNA–TBP–TFIIA–TFIIB–Tfg2 linker (Extended Data Fig. 7b), TFIIE–Tfg2 WH (Extended Data Fig. 7c), TFIIF (Extended Data Fig. 7d), and TFIIF dimerization domain (Extended Data Fig. 7d). Each class was refined using the 3D auto-refine procedure against the respective particles within that class with a soft reference mask in the shape of the OC (maximum diameter of 270 Å), generated in RELION58. The OC1 reconstruction (improved Pol II core, B-ribbon, and E-ribbon density) was determined from 102,876 particles to a resolution of 3.58 Å and with a temperature factor of −111 Å2 (Extended Data Fig. 8a). The OC2 reconstruction (improved upstream DNA, TBP, TFIIA, TFIIB, and Tfg2 linker density) was determined from 17,282 particles to a resolution of 3.97 Å and with a temperature factor of −95 Å2 (Extended Data Fig. 8b). The OC3 reconstruction (improved TFIIE density) was determined from 11,231 particles to a resolution of 4.35 Å and with a temperature factor of −125 Å2 (Extended Data Fig. 8c). The OC4 reconstruction (improved TFIIF dimerization and Tfg1 arm density) was determined from 29,455 particles to a resolution of 3.89 Å and with a temperature factor of −79 Å2 (Extended Data Fig. 8d). Focused refinement of the upstream DNA assembly (OC2-focused) was achieved by continuing the auto-refinement (round 1, class 4; Extended Data Fig. 7c) from the first round of local searches using a corresponding soft mask. Focused refinement of TFIIE–Tfg2 WH (OC3-focused) was achieved by continuing the OC3 auto-refinement from the first round of local searches using a soft mask encompassing TFIIE, Tfg2 WH, and the interacting segment of upstream DNA. Focused refinement of TFIIF dimerization domain (OC4-focused) was achieved by continuing the auto-refinement (round 2, class 2; Extended Data Fig. 7d) from the first round of local searches using a soft mask encompassing the TFIIF dimerization domain and the Pol II lobe. The resolution of focused refinements was determined using a soft mask with a 30-pixel soft edge62, to 4.7 Å, 7.5 Å, and 4.09 Å for OC2-, OC3-, and OC4-focused refinements respectively (Extended Data Fig. 8b, c, d).
Single particle cryo-EM analysis of the closed complex was carried out essentially as for the open complex, with the following differences. Reference-free 2D classification of 10,591 particles, picked semi-automatically using e2boxer.py from EMAN256, gave three representative 2D class averages that were low-pass filtered to 30 Å resolution and used as templates for automated particle picking in RELION of all micrographs60. The resultant 155,079 particles were screened manually and by reference-free 2D classification, yielding 111,625 particles that were used for subsequent processing. The cITC (EMD-2785), low-pass filtered to 50 Å, was used as the reference model for the initial 10,591 particle set, and the resultant 3D reconstruction (14 Å resolution), again low-pass filtered to 50 Å, was used as initial reference model for 3D refinement using the full cryo-EM data set. This revealed the CC at 7.5 Å, and was improved by particle polishing to 6.5 Å resolution using RELION 1.4 beta58. Hierarchical 3D classification without image alignment using a soft mask encompassing the complete CC resulted in two populations, CC and OC5, respectively (Extended Data Fig. 7e). Both classes were refined using the 3D auto-refine procedure against the respective particles within that class with a soft mask in the shape of the CC. The CC soft mask was also suitable for the OC as both complexes have a similar shape. The CC reconstruction was determined from 7,527 particles to a resolution of 8.2 Å. The OC5 reconstruction was determined from 79,797 particles to a nominal resolution of 6.1 Å and a temperature factor of −176 Å2 was applied (Extended Data Fig. 8g). To improve the densities TFIIE, Tfg2 WH and downstream DNA in the CC, we carried out focused classification of the Pol II stalk and subsequently TFIIE, Tfg2 WH and downstream DNA using soft masks encompassing these regions. The individual classes were refined as before and yielded a CC at 8.8 Å comprising 5,690 particles and a temperature factor of −300 Å2 was applied (Extended Data Fig. 8f). A similar focused classification scheme for OC5 revealed moderately improved density for TFIIE (not shown).
Local resolution estimates were determined using a sliding window of 403 voxels as previously described except that a single pair two half-maps was used and resolution estimates were not capped at the nominal resolution19 as no local filters were applied.
A composite model of the OC was obtained using cryo-EM densities OC1–OC4 and the focused refinements of OC2, OC3, and OC4 particles. Structural models were built in COOT63 unless indicated otherwise. Models were refined using the real space refinement routine in Phenix64 into the respective OC density, as indicated, with secondary structure and rotamer restraints. First, structural models of Pol II (lacking Rpb4–Rpb7), TFIIB B-ribbon (residues 22–59), and downstream DNA (PDB: 4V1N19) were placed into the OC1 map using UCSF Chimera61, followed by rigid-body group refinement in Phenix64. Models of the Pol II core and TFIIB B-ribbon were adjusted and extended manually, and refined in Phenix64 into the OC1 density. The Rpb4–Rpb7 structure (PDB: 4V1N) was fitted, and residues at the base of the stalk (Rpb7 1–81, 150–159) were modified to fit the density and refined into the OC1 map. TFIIE was modelled using structural information on eWH33, E-ribbon34, and WH136, and a homology model for WH2 (Fig. 3a, b). A homology model of the human TFIIEα E-ribbon (PDB: 1VD4 (ref. 34)) was generated using MODELLER65 (Tfa1 residues 121–158) and rigid-body fitted into the unsharpened OC1 density. Upstream DNA (PDB: 4V1N), TFIIB N-terminal cyclin (PDB: 4BBR, chain M and residues 122–213), the homology model of TFIIB C-terminal cyclin domain (PDB: 4V1N, chain M and residues 233–343) were fitted into the OC2 cryo-EM density. Models of Pol II protrusion, wall and the TFIIB N-terminal cyclin were adjusted and extended manually, and the TFIIB C-terminal cyclin (residues 236–328) and Tfg2 linker regions (residues 249–280) were built into OC2 and OC2-focused maps and refined into the OC2 map in Phenix64. X-ray structures of TBP (PDB: 1YTB66, chain A), and TFIIA (PDB: 1YTF25, chains B–D) were individually fitted into the OC2 map obtained from focused refinement (OC2-focused). The fit of upstream DNA, from the upstream edge of the DNA bubble to TBP, was improved in UCSF Chimera61 using the OC3 map. Protein models of Tfa1 eWH (residues 1–90) and Tfa2 WH2 (residues 187–249), generated with the I-TASSER prediction server67, were fit into the OC3 density and adjusted. The TFIIE linker helices were built de novo and modified to match the density, and together with Tfa1 eWH and Tfa2 WH2 models were refined into the unsharpened OC3 map in Phenix64 and subjected to the phenix.model_idealization routine to optimize geometry. Published NMR data of the Tfg2 WH domain68 (BMRB accession number 17916) was used to calculate a low-energy model with the BMRB CS-ROSETTA server69. The Tfg2 WH and an I-TASSER model of Tfa1 WH1 (residues 123–181) were placed into the OC3 density obtained from focused refinement (OC3-focused). The Tfg2 WH positioning is also consistent with NMR data for the Tfg2 WH–DNA interface68. A part of the Tfg1 arm domain (residues 327–349) was built into OC3 and OC4 densities and refined into the OC4 density. A previous homology model of the TFIIF dimerization domain18 was used as a starting template for modelling into OC4 and OC4-focused density maps, and was refined into the OC4 density. The crystal structure of the Tfg1 N-terminal peptide (residues 21–35, see below) was fitted into an OC4 density (round 2 class 2, Extended Data Fig. 7d). The individually refined models showed good stereochemistry and were validated with Molprobity70, and the FSC of map versus model (Extended Data Figs 8 and 9).
To generate the CC model, the OC model of Pol II, TFIIA, TFIIB, TBP, TFIIE E-ribbon and TFIIF dimerization domain was rigid-body fitted into the CC density using an automated global 6D correlation search in Situs71. The remaining protein part was divided into two rigid bodies containing (i) Tfa1 and Tfa2 WH2, and (ii) Tfa2 WH1 and Tfg2 WH, which were independently fitted into the CC density using Situs71. To model closed promoter DNA, upstream DNA was extended with canonical duplex B-form DNA in COOT63 and rigid-body fitted in UCSF Chimera57 to reflect the density.
All figures were generated using UCSF Chimera61.
Crystallographic analysis of Pol II–TFIIF complex
The structure of the Tfg1 N-terminal region (residues 21–35) bound to Pol II was determined by X-ray crystallographic analysis. 12-subunit Pol II was prepared and crystallized53, and TFIIF or selenomethionine (SeMet)-substituted Tfg1 peptide (residues 19–41, Peptide Speciality Laboratories GmBH, Heidelberg) was added to the cryo-protectant as described72. Diffraction data were collected on a PILATUS 6M detector at the X06SA beamline (SLS, Villigen, Switzerland) at 100 K. Data for the Pol II–Tfg1 SeMet peptide crystal was collected at a wavelength of 0.97972 Å and for the Pol II–TFIIF crystal at 0.91889 Å. Data were processed with XDS and XSCALE73. The structure was phased with the crystal structure of the 12-subunit Pol II (PDB: 3PO2) lacking nucleic acid and refined using BUSTER74. Model building of the TFIIF N-terminal residues in COOT63 was guided by a selenium anomalous difference Fourier peak (M27). Subsequent refinement in BUSTER used secondary structure restraints for the Tfg1 peptide helix. The final structures had an Rfree factor of 18.0% and 19.4% for the Pol II–Tfg1 SeMet peptide crystal and the Pol II–TFIIF crystal, respectively (Extended Data Fig. 9c), and showed good stereochemistry70. In the Pol II–Tfg1 SeMet peptide structure 90% of the residues fall in favoured regions of the Ramachandran plot and 3% in disallowed regions. For the Pol II–TFIIF structure 89% of the residues fall in favoured regions of the Ramachandran plot and 3% in disallowed regions.
Yeast and functional assays
TFIIE interfaces with Pol II and DNA were probed by in vivo mutagenesis. The yeast strain used for TFA1 genetic assays16 was provided by S. Hahn (Fred Hutchinson Cancer Research Center). Mutations were introduced into a plasmid encoding TFA1 (pSH810, ARS CEN LEU2 3×Flag), as indicated in Extended Data Fig. 3g. The tfa1(ΔE-wing) mutant was generated by deletion of TFA1 residues 71–84, and their replacement by the residues GSG. The TFA1 constructs were transformed into the shuffle strain, and were streaked twice on −Ura −Leu plates, and subsequently onto yeast extract-peptone-dextrose (YEPD) plates. Yeast were freshly grown in YEPD medium and resuspended in water to an OD of 1 at 600 nm, and tenfold dilutions were spotted on 5-fluoroorotic acid (5-FOA) and YEPD plates, and incubated at 30 °C.
To further characterize the interfaces between TFIIE and the initiation complex, recombinant TFIIE mutants were purified according to the same protocol as wild-type TFIIE (Recombinant proteins, Extended Data Fig. 3g, h). The Tfa1(poly-Ala E-wing) mutant was generated by replacement of TFA1 residues 71–84 with poly-Alanine.
To assess the interaction of wild-type TFIIE and the TFIIE mutants with the CC by protein pulldown, 3 μg purified Pol II was first biotinylated on the Rpb3 subunit as described19. The CC, containing biotinylated Pol II, was subsequently prepared as above (‘preparation of initiation complexes’), but without TFIIE. This preparation was immobilized on 15 μL Dynabeads M280 streptavidin resin (Life Technologies), equilibrated in buffer Q. 10 μg TFIIE or TFIIE mutant (tenfold molar excess over Pol II) was incubated with the immobilized CC or control beads for 1 h at 4 °C. The beads were washed four times and bound proteins were analysed by SDS–PAGE (Extended Data Fig. 3h).
Promoter-dependent in vitro transcription was used to determine the activity of TFIIE and the TFIIE mutants. The yeast strain used for TFA1 genetic assays16 was transformed with a plasmid containing 3×Flag-tagged TFA1 (pSH810, ARS CEN LEU2 3×Flag). Transformants were streaked once onto −Ura −Leu plates, once onto −Leu plates, twice onto 5-FOA plates, and subsequently onto YEPD plates. Nuclear extract from 3 l yeast culture was prepared as described19. The nuclear extract was immunodepleted of 3×Flag–Tfa1 as described19 with the following modifications. Before nuclear extract was incubated with anti-Flag M2 agarose beads (Sigma), beads were incubated with 1 mg ml−1 bovine serum albumin (BSA) protein (Sigma) for 1 h at 4 °C on a turning wheel followed by three wash steps. Immunodepleted nuclear extract was separated from beads by Micro Bio-Spin chromatography columns (Biorad). Specificity of the depletion was confirmed by western blot carried out as described previously19 for 3×Flag-tagged Tfa1 (Macs Miltenyi Biotec, 130-101-572), Rpb3 (Neoclone, WP012), TFIIB (Abcam, sc-274) and Histone H3 (Abcam, ab21054) (Extended Data Fig. 3i). The secondary antibodies anti-rabbit IgG horse-radish peroxidase (HRP; GE Healthcare, NA934) and anti-mouse IgG HRP (Abcam, ab5870) were used (Extended Data Fig. 3i). Activator- and promoter-dependent in vitro transcription and primer extension were carried out as described previously19. Recombinant TFIIE (5 pmol) and TFIIE mutants (5 pmol) were added to the depleted nuclear extract as indicated in Fig. 3c. Transcripts were visualized on a denaturing 8% polyacrylamide TBE gel with a Typhoon 9500 scanner (GE Healthcare) and quantified with ImageQuant (GE Healthcare). For quantification, the relative activity of each variant compared to TFIIE was calculated for each replicate. The mean intensity and standard deviation of three replicates was calculated from their relative activities. A second RNA product from the HIS4 promoter was observed, apparently resulting from an alternative upstream transcription start site. Although some differences in the relative use of the two TSSs are observed in the assay, we refrained from interpreting these.
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We thank C. Bernecky, W. Mühlbacher, S. Neyer, S. Sainsbury, and D. Tegunov for help and discussions; L. Larivière and L. Wenzeck for cloning and initial purification of TFIIE; W. Mühlbacher for initial cloning of TFIIA; S. Bilakovic for the modified pET-DUET-1 vector; J. Mahamid for help with data collection for the CC; K. Maier for help with yeast growth assays; M. Raabe and H. Urlaub for protein identification; K. Kinkelin for initial Pol II–TFIIF co-crystallization; and S. Hahn for providing the TFA1 yeast strain and the shuffle plasmid pSH810. C.P. (SFB860), M.H. (GRK1721), and P.C. were supported by the Deutsche Forschungsgemeinschaft, the Advanced Grant TRANSIT of the European Research Council, and the Volkswagen Foundation.
Extended data figures and tables
Extended Data Figures
- Extended Data Figure 1: Modelling of open complex cryo-EM densities. (846 KB)
a, SDS–PAGE analysis of OC–cMed–Med1 complex after size-exclusion chromatography. Protein colours as in Fig. 1. Although core Mediator was required for stable association of TFIIE, it largely dissociated under cryo-EM conditions as observed previously19. Some remaining core Mediator was flexible and located as described previously19, but could not be included in further high-resolution analysis. b, Cryo-EM micrograph of the OC–cMed–Med1 complex. Scale bar, 50 nm. c, Ten representative reference-free 2D class averages of OC–cMed–Med1 reveal flexibility of the upstream DNA assembly including TFIIE (green arrow) and very weak density for core Mediator (orange arrow). Compare Extended Data Fig. 7a, c. d, Composite cryo-EM density of the OC shown in front and top views50. Colours indicate the cryo-EM densities used for modelling of the open complex (OC1, grey; OC2, green; OC2-focused, yellow; OC3, salmon; OC3-focused, blue; OC4 purple; OC4 round 2 class 2, light blue). Shown are the unsharpened cryo-EM densities. The percentage of particles from the full set of 257,259 that was used for the respective reconstruction is indicated. e, Composite cryo-EM density of the OC superimposed on a ribbon model of the OC, coloured as in Fig. 1. The composite cryo-EM density enabled modelling of the initiation factors and DNA. Our structure also enabled correction of the revised yeast initiation complex model obtained by Murakami et al. from cryo-EM at 6 Å resolution21, and we note the following differences between the structures, superimposed on Rpb1: (1) The TFIIF Tfg2 WH domain is rotated by ~180°, which is further inconsistent with nuclear magnetic resonance (NMR) data on the TFg2 WH–DNA interface68 and fits comparatively worse to protein–protein crosslinking data between the Tfg2 WH and Tfa2 WH1 (ref. 17). (2) Domains of TFIIE, except Tfa2 WH1, were placed incorrectly: Tfa1 eWH (rotation and translation into the E-linker density; 17 Å distance for helix α3 in our CC), Tfa1 E-ribbon (rotation and translation into E-linker density; 35 Å distance between the Zn atoms), and Tfa2 WH2 (~180° rotation). Further, the Tfa2 E-tether region was incorrectly assigned to density belonging to the Tfa1 eWH. The Tfa1 E-linker was not modelled. (3) The TFIIF Tfg1 arm was modelled into an empty space lacking density, and the Tfg1 helix α0 was absent. Our models of the TFIIF dimerization domain, Tfg2 linker, Tfg1 N terminus, and Tfg1 arm fit into densities from a recent study21, indicating the electron microscopic reconstruction is correct, but that the modelling was premature at the available resolution. f, Ribbon model of the OC coloured according to how different parts of the OC were modelled into the OC cryo-EM densities (see d). Regions with atomic (light blue) and backbone models (orange), and DNA (dark blue) are indicated. Views as in d. g, Representative regions of the sharpened cryo-EM densities OC1 (3.6 Å), OC2 (4.0 Å), and OC4 (3.9 Å) are shown with the underlying refined coordinate model. The OC1 density shows clear side-chain features for Rpb1 clamp helices α8 and α9 and Rpb2 β33, the OC4 density for Tfg1 β2 that is part of the dimerization domain, and the OC2 density for part of the Tfg2 linker. For OC nomenclature, see Extended Data Fig. 7. h, Fit of the TBP crystal structure (PDB: 1YTB)66 to the OC2 cryo-EM density, shown in a Pol II side view50. i, Fit of TFIIB N- and C-terminal cyclin domains, B-linker and B-reader, and B-ribbon elements to OC1 and OC2 cryo-EM densities. The B-linker element displays weak density, and the B-reader is not observed. j, Fit of the TFIIA crystal structure (PDB: 1YTF)23 to OC2-focused cryo-EM density in a Pol II top view50 (left). The four-helix bundle undergoes a minor rotation towards the β-barrel, and is apparently flexible (compare Extended Data Fig. 5e). Toa1 (middle) and Toa2 (right) subunit structures are shown. A large non-conserved insertion in Toa1 (Δ95–209), lacking in recombinant TFIIA (Methods), may affect the relative positioning of the four-helix bundle to the β-barrel. k, Fit of the TFIIF model to OC cryo-EM densities viewed from the top50. TFIIF dimerization domain and Tfg1 N-terminal region, arm, and charged helix elements are superimposed on the OC4 cryo-EM density. Tfg2 linker and WH domains are superimposed on OC2 and OC3-focused cryo-EM densities, respectively. Subunit architectures for Tfg1 (middle) and Tfg2 (right) subunits are shown, indicating disordered regions. Secondary structure elements were labelled according to the crystallographic model of the human RAP30–RAP74 heterodimer75. l, Fit of the TFIIE model to OC cryo-EM densities shown from the front50 (left). Models for Tfa1 eWH, E-linker and E-ribbon are superimposed onto OC1 and OC3 densities. Models for Tfa2 WH1 domain, Tfa2 WH2 and E-tether were fitted into OC3-focused and OC3 densities. Tfa1 (middle) and Tfa2 (right) subunits are shown, indicating disordered regions. The connectivity of the E-tether helices remains uncertain. m, Fit of promoter DNA to OC cryo-EM densities is shown in a side view50. A weak density for single-stranded template DNA contacts the Pol II fork loop 1, and is indicated by a blue arrow. Upstream and downstream DNA models are superimposed with OC3 and OC1 densities, respectively. The location of the Pol II active site magnesium ion is indicated.
- Extended Data Figure 2: Details of TFIIF and the upstream DNA assembly. (1,199 KB)
a, View of the open complex from the side50. Pol II elements external 2 (dark green), lobe (yellow), protrusion (orange), Pol II subunit Rpb12 (dark blue) and basal factors TBP, TFIIB, and TFIIF are coloured as in Fig. 1. The remainder of the open complex is transparent. Green and purple boxes indicate the locations of TFIIB C-terminal cyclin and TFIIF dimerization domains, respectively. b, Interactions of TFIIB C-terminal cyclin domain with Pol II protrusion, Rpb12, Tfg2 linker and DNA. Colours as in a. c, Details of TFIIF dimerization domain interactions with Pol II external 2 and lobe50. d, Crystallographic analysis of the yeast-specific Tfg1 N-terminal region. Weak density for the Tfg1 N-terminal region was observed by cryo-EM (OC4 round 2 class 2) at low contour level (0.0155) close to Pol II elements external 1 and the hybrid binding region50 (left). X-ray analysis (right) of the corresponding peptide (Tfg1 F21–R35) enabled modelling and assignment of residue M27 (indicated with asterisk) owing to the anomalous signal. The Fo − Fc electron density map (grey, contour level 2.5σ), seleno-methionine anomalous difference Fourier (yellow, contour level 5σ), and final model in ribbon presentation (purple) are shown. The sequence of the synthetic peptide used for soaking into Pol II crystals is shown below. The modified methionine residue and predicted secondary structure are indicated. e, The Fo − Fc electron density maps obtained from soaking Pol II crystals with TFIIF (purple) and seleno-methionine labelled peptide (grey), respectively, show similar density in the same location on Pol II. f, The putative Tfg2 C terminus contacts TBP. Viewed from the side50. A tubular cryo-EM density from the OC3 map, low-pass filtered to 8 Å, emanates from the TFIIF Tfg2 WH–TFIIE Tfa2 WH1 density, and was tentatively assigned to the Tfg2 C-terminal region. The putative Tfg2 density reaches the TBP subunit, consistent with their suggested interaction29, 76.
- Extended Data Figure 3: Structure–function analysis of TFIIE and its interactions in the open complex. (598 KB)
a, The architectural model of TFIIE contains all regions required for viability in yeast16. A domain schematic (top) indicates the good overlap between modelled (dashed line) and functionally essential regions. Essential (grey), partially redundant Tfa2 WH1 and WH2 domains (blue), and non-essential elements (cyan) are indicated on the TFIIE model, shown in previously defined front and top views50 of Pol II. b, TFIIE sequence conservation. The sequence conservation among Saccharomyces cerevisiae, Schizosaccharomyces pombe, Drosophila melanogaster, Gallus gallus, and Homo sapiens was mapped onto a ribbon representation of the TFIIE model. Highly, strongly, weakly and non-conserved residues are coloured in green, yellow, white, and grey, respectively. The location of a non-modelled helical density in the OC3 cryo-EM map, which may correspond to Tfa1 helix α7, is indicated. Views as in a. c, An additional density (green) in the OC3 cryo-EM map on top of the Tfa1 E-wing was tentatively assigned to Tfa1 helix α7 and this may stabilize the long β-hairpin. A front view is shown50. d, Tfa1–FeBABE cleavage sites in TFIIE16 are consistent with the TFIIE architecture. e, Tfa1– and Tfa2–FeBABE cleavage sites in the Pol II clamp16 and a protein–protein crosslink between Rpb1 K212 (Pol II clamp)–Tfa2 K277 (TFIIE E-tether)17 are consistent with the location of eWH and E-tether. f, Tfa2–Tfg2 protein–protein crosslinks17 are consistent with the Tfg2WH–Tfa2 WH1 architecture. g, The TFIIE mutations used for functional characterization were mapped onto a domain schematic and the model of TFIIE, shown in a front view50. h, Pulldown assays with recombinant TFIIE variants carrying mutations at the TFIIE–CC interface revealed that the E-ribbon is essential for TFIIE recruitment. For details of the TFIIE mutants, see g. Pulldowns were analysed by SDS–PAGE (Coomassie staining). To confirm the integrity of the purified TFIIE variants, 2 μg were analysed (left). Some minor contaminant and degradation bands of TFIIE are indicated by an asterisk. The bead elution from the pulldown assay is shown (middle), providing negative (no TFIIE) and positive (TFIIE) controls in the two leftmost lanes. The binding of all TFIIE variants to the CC was impaired compared to the wild-type protein, with the exception of the Tfa1(ΔE-wing) mutant, suggesting that all other interfaces contribute to TFIIE binding affinity. The most severe binding defect was observed upon mutation of three residues in the E-ribbon (Tfa1(L134/V137/L140)) to glutamate or alanine. This suggests that the E-ribbon is largely responsible for recruitment of TFIIE to the CC. The bead-only control (right) indicated that TFIIE and TFIIE variants did not show unspecific binding to the beads. i, Western blot analysis of the 3×Flag-tagged Tfa1 confirms specific immune-depletion of Tfa1 in the nuclear extract (NE), whereas levels of Pol II (Rpb3), TFIIB, and Histone H3 were unaffected. j, Yeast complementation assays were performed in triplicate experiments with wild-type TFA1, an empty vector, and TFA1 variants with mutations in the TFIIE eWH domain (N50E/K51E/T52E, N50A/K51A/T52A, and P56A/A59E/R62E in eWH helix α3, and ΔE-wing), or the E-ribbon(L134E/V137E/L140E) (see Methods). k, The long E-wing in the TFIIE subunit Tfa1 eWH is characteristic of WH domains involved in DNA strand separation77. The upstream edge of the transcription bubble and eWH domain are shown in a front view50 rotated by ~20° in the horizontal axis. Corresponding regions of human (Hs) Werner syndrome ATP-dependent helicase (WRN) WH (PDB: 2WWY) and RecQ1 WH (PDB: 3AAF) domains are shown.
- Extended Data Figure 4: Closed complex and spontaneously formed open complex. (619 KB)
a, SDS–PAGE analysis of CC–cMed–Med1 complex after size-exclusion chromatography. Protein colours as in Fig. 1. b, Cryo-EM micrograph of the CC–cMed–Med1 complex. Scale bar, 50 nm. c, Ten representative reference-free 2D class averages of CC–cMed–Med1 reveal flexibility for the upstream complex. Core Mediator was not retained during cryo-EM analysis. d. Detailed view of the Pol II funnel helices in the CC (top) and OC5 (bottom) densities. e, Promoter sequences and differences in protein–DNA interactions are shown for the two distinct nucleic acid scaffolds used for preparation of closed and open complexes (compare Fig. 1d). Coloured bars indicate DNA–protein interaction. Solid, shaded, and empty circles respectively represent nucleotides included in the structure, excluded owing to weak cryo-EM density, or excluded owing to absence of cryo-EM density. Analogous yeast (black) and human (grey) numbering of promoter DNA is shown. The TATA-box sequence (red box) and HIS4-promoter sequence absent in the modified OC nucleic acid scaffold19 (grey box) are indicated. Protein–DNA interactions in the region covered by the light grey box are unchanged between CC and OC, and shown only for the OC for clarity. Unique and altered interactions are shown for each complex. DNA–TFIIEα photo-crosslinks, indicated by black asterisks, were observed in a closed but not open promoter state40 and are consistent with the CC model. f, Fit of TFIIE, Tfg2 WH and downstream DNA into CC density. Two rigid bodies were used for fitting: (i) Tfg2 WH and Tfa2 WH1 and (ii) Tfa2 WH2, eWH, E-linker and E-tether helices. Although the overall fit reflects density well, the eWH domain and its E-wing may be rotated further away from promoter DNA. g, Details on the location of downstream DNA (template, blue; non-template, light blue), Tfg2 WH, and Tfa2 WH1 and WH2 in the closed (dark colours) and open (light colours) complexes in the same view as in f. h, Cryo-EM density of OC5 and the OC ribbon model are shown in a front view50. The OC5 map shows weak density in regions of upstream assembly, TFIIE, and DNA that may be caused by increased flexibility owing to the heterogeneous population of spontaneously opened DNA. Colours as in Fig. 1. i, Fit of promoter DNA to cryo-EM densities of CC and OC5, shown in a side view50.
- Extended Data Figure 5: Pol II cleft clearance, structural flexibility and rearrangements in the OC. (389 KB)
a, Pol II lid and fork loop 1 assume new conformations in the OC, clearing the Pol II cleft for loading of single-stranded template DNA. Arrows indicate the direction of movement of the two Pol II elements, and the template DNA loading path. The lid (dark red) in the open complex is moved in comparison to the lid of a Pol II–TFIIB ITC crystallographic study (PDB: 4BBS). Yellow and red boxes indicate zoomed-in regions of b and c, respectively. b, The movement in the Pol II lid leads to a steric clash with the TFIIB B-reader, observed in a Pol II–TFIIB ITC crystal (PDB: 4BBS), and facilitates its withdrawal in the open complex. In particular the lid residue F252 clashes with W63 and S67 of the B-reader. The OC1 cryo-EM density is shown for both lid and B-reader elements. c, The cryo-EM density of the OC1 reveals an ‘open’ Pol II fork loop 1 and a stably associated fragment of putative template DNA. The ‘open’ state of fork loop 1 provides additional space for loading of single-stranded template DNA past the Pol II rudder, towards the active site cleft. d, The position of the TFIIB N-terminal cyclin domain (light green) is altered in comparison to a Pol II–TFIIB ITC crystal structure15 (dark grey), but similar to its location in a cITC19 (light grey), probably owing to the presence of DNA. e, Flexibility of the upstream DNA assembly. The cryo-EM data of the OC was sorted on the basis of structural differences using an upstream assembly mask that included upstream DNA, TFIIA, TBP, and TFIIB cyclin domains (OC2 round 1, compare Extended Data Fig. 7c). Four of five resultant classes revealed different positions of the upstream complex, indicated here by fitted ribbon models of the OC. Previously defined front and side views50 are shown. Class 2 (middle) revealed the TFIIA four-helix bundle rotated by 85°, consistent with a high degree of flexibility. Class 4 represents the largest fraction of the data (31%), and gave a more defined density for the upstream complex, which was improved by further classification (Extended Data Fig. 7c). Class 5 presented with no density for the upstream complex or the Tfg2 linker, but did show density for the TFIIB B-ribbon and the TFIIF dimerization domain, suggesting that TFIIB and TFIIF remained bound to the complex. This is consistent with TFIIF-dependent association of the TFIIB-core domain with the Pol II wall27, and this apparently requires an ordered Tfg2 linker. f, The Rpb4–Rpb7 stalk adopts different positions in cITC, cITC-cMed, and OC. This suggests that Mediator and TFIIE may bind co-operatively. This is consistent with previous findings78 and with pulldowns (Extended Data Fig. 3h), which suggest that the TFIIE E-ribbon–stalk interface, which is important for TFIIE recruitment, is stabilized in the presence of Mediator.
- Extended Data Figure 6: Pol II clamp positions and TFIIB B-reader mobility during DNA opening. (408 KB)
a, The yeast CC is shown from a side view50, indicating the path of DNA and location of TFIIE. The eye symbol (grey) indicates the point of view in b. b, The Pol II clamp may undergo transitions during DNA opening as indicated. The OC model of the Pol II clamp is shown superimposed on yeast CC (this study), and yeast OC (this study). The OC model Pol II clamp was rigid-body fitted to the human CC cryo-EM density20 (EMD-2306) and is superimposed. The view is from the front50. c, The TFIIB B-reader element shows strong density only in the ITC state, suggesting that its mobility in earlier states may be important for maintaining a cleared path for template DNA loading into the Pol II cleft. Ordering of the B-reader may further lead to stabilization of the upstream promoter assembly that is flexible in the OC (Extended Data Figs 5e, 7c). Cryo-EM densities for yeast CC (this work), OC5 (this work), OC (this work), and ITC (EMD-2785) complexes are superimposed on the TFIIB model (PDB: 4BBS for the B-linker and B-reader). As secondary structure elements could not be resolved in the human CC20, we excluded this cryo-EM density from comparison.
- Extended Data Figure 7: Three-dimensional classification of cryo-EM data. (454 KB)
a, Three-dimensional image classification of the cryo-EM data set into eight classes using an initial OC reconstruction as the reference model, revealed heterogeneity. The percentage of single particles contributing to each class is provided. To help visualize structural differences, 3D reconstructions of the OC are coloured according to mobile regions: Pol II core, TFIIB B-ribbon (grey); upstream DNA, TFIIA, TBP, TFIIB cyclin domains, Tfg2 linker (green); TFIIF dimerization domain (purple); TFIIE except E-ribbon, Tfg2 WH (magenta); Pol II Rpb4–Rpb7 stalk and E-ribbon (blue); cMed–Med1 (yellow). b, Focused classification into five classes using a mask covering the Pol II stalk and E-ribbon. The resultant class 1 (OC1) was subsequently refined to 3.58 Å resolution (grey box) and revealed the location of the TFIIE E-ribbon. Colours as in a. c, Improvement of densities for Tfg2 linker, TFIIB, and TFIIE, through rounds of focused 3D classification using various masks. First, heterogeneity due to flexibility of upstream DNA and associated factors was overcome by applying a mask around this region (round 1). Focused refinement of the upstream DNA assembly of the resultant class 4 of round 1 (OC2-focused), improved the density quality for TFIIA (Extended Data Fig. 1j). Classification of the OC2-focused density revealed the upstream DNA complex (OC2) at 3.97 Å resolution (green box). Separate classification of class 4 of round 1 using OC, Pol II stalk and TFIIE E-ribbon, and TFIIE masks yielded class 1 of round 4 (OC3, magenta box) that contained a complete TFIIE density at a nominal resolution of 4.35 Å after 3D refinement (see Extended Data Fig. 8c). The small fraction of stably bound TFIIE is consistent with its reduced affinity to the pre-initiation complex79. Focused refinement of OC3 with a TFIIE–stalk mask (OC3-focused) improved density for Tfg2 WH and Tfa2 WH1 domains. Colours as in a. d, To improve the density of TFIIF dimerization domain and the Tfg1 arm, three rounds of classification using a TFIIF, TFIIF dimerization domain, and OC mask were employed. Class 2 of round 2 (cyan box) enabled fitting of the Tfg1 N-terminal peptide, which was resolved by X-ray analysis (Extended Data Fig. 2d, e). This class was further refined locally using a mask covering the TFIIF dimerization domain, and then classified with an OC mask, revealing class 6 of round 3 at 3.89 Å resolution after 3D refinement (purple box). Colours as in a. e, 3D classification of the CC cryo-EM data set into four classes, using an initial CC reconstruction as the reference model, revealed heterogeneity. Mobile regions in the reconstructions are highlighted: promoter DNA (blue), TFIIE (except E-ribbon), and Tfg2 WH (orange). Classifying the most populated classes from round 1 into three classes unexpectedly revealed open and closed promoter DNA states in the data set: CC (round 2, class 1) and OC5 (round 2, class 3). Class 3 of round 2 (OC5) was refined to 6.1 Å resolution (blue box). Class 1 from round 2 was further classified into three classes. The resultant class 3 of round 3 revealed density for closed downstream promoter DNA above the Pol II cleft, and TFIIE. The cryo-EM density for downstream DNA and TFIIE was improved by focused classification using two soft-edged masks. A mask covering the Pol II Rpb4–Rpb7 stalk yielded a class with better occupancy for the stalk (round 4, class 3), which was further sorted using a mask covering TFIIE and Tfg2 WH to improve their densities. Class 1 of round 5 was refined to 8.8 Å resolution (CC, orange box).
- Extended Data Figure 8: Resolution of cryo-EM reconstructions. (837 KB)
a, Gold-standard FSC (left) of the OC1 cryo-EM single particle reconstruction (FSC = 0.143). Orientation distribution plot of all particles that contribute to the OC1 reconstruction (middle). The OC1 cryo-EM map is shown (right) from a front view50 and a central slice through the reconstruction, which are coloured by local resolution as described19. b, As in a but for the OC2 reconstruction. The gold-standard FSC for the density obtained from focused refinement (OC2-focused) with a soft mask around the upstream DNA assembly is indicated in grey (see Methods). The region masked for focused refinement is indicated with a grey outline on the cyro-EM map coloured by local resolution (right). c, As in b, but for the OC3 and OC3 focus-refined reconstructions. d, As in a, but for the OC4 and OC4 focus-refined reconstructions. e, As in a, but for the CC reconstruction. f, As in a, but for the OC5 reconstruction.
- Extended Data Figure 9: Data collection, refinement statistics, and structure validation. (209 KB)
a, Cryo-EM data collection and refinement statistics of the OC structure. Different regions of the composite OC structure were refined into OC1, OC2, and OC4 maps as described (see Methods) to obtain an atomic model for 90% of the structure. b, Gold-standard FSC between the respective coordinate models and local regions of the OC1, OC2, and OC4 cryo-EM maps used for model refinement and between overall OC and CC models compared to OC3 (best TFIIE density) and CC cryo-EM maps. c, X-ray crystallographic data collection and refinement statistics.