www.sciencemag.org/cgi/content/full/science.1238724/DC1

Supplementary Material for

Architecture of an RNA Polymerase II Transcription Pre-Initiation Complex

Kenji Murakami, Hans Elmlund, Nir Kalisman, David A. Bushnell, Christopher M. Adams, Maia Azubel, Dominika Elmlund, Yael Levi-Kalisman, Xin Liu, Michael Levitt,

Roger D. Kornberg*

*Corresponding author. E-mail: kornberg@stanford.edu

Published 26 September 2013 on Science Express

DOI: 10.1126/science.1238724

This PDF file includes:

Materials and Methods

Supplementary Text

Figs. S1 to S14

Tables S4 and S5

References (46–63) Other Supplementary Material for this manuscript includes the following: (available at www.sciencemag.org/cgi/content/full/science.1238724/DC1)

Tables S1 to S3 as Excel files Movies S1 to S3 Protein Data Bank file for the PIC model (.pdb file)

Supplementary Materials for

Architecture of an RNA polymerase II transcription pre-initiation complex

Kenji Murakami, Hans Elmlund, Nir Kalisman, David A. Bushnell, Christopher M. Adams, Maia Azubel, Dominika Elmlund, Yael Levi-Kalisman, Xin Liu, Michael Levitt, Roger D.

Kornberg correspondence to: kornberg@stanford.edu

This PDF file includes:

Materials and Methods Supplementary Text Figs. S1 to S14 Captions for Tables S1 to S5 Captions for Movies S1 to S3

Other Supplementary Materials for this manuscript includes the following:

Tables S1 to S5 Movies S1 to S3 Protein Data Bank file for the PIC model (file name: Murakami_et_al_PIC_model.pdb)

Materials and Methods PIC reconstitution on a preparative scale TFIIA, TFIIB, TBP, TFIIE were available in recombinant form (21). Full-length S. cerevisiae TFIIS was subcloned into pRSFDuet vector with N-terminal His6-tag and a linker of a tobacco etch virus (TEV) protease site, and overexpressed in Rosetta 2(DE3) (Novagen). The cells were lysed by sonication, and the soluble fraction was purified using Ni2+-affinity chromatography. The Ni-column eluate was incubated with TEV protease for 15 h at 4°C, and was further purified by using Ni, Uno-S (Bio-Rad), and Superdex 200 columns (GE Healthcare). Pol II-TFIIF complex was isolated from yeast, as previously published (21). 10-subunit TFIIH was isolated from yeast tfb6Δ strain harbouring TAP-tagged Tfb3 as published before (20, 21). TFIIH lacking TFIIK was isolated from Tfb4-TAP tagged strain with minor modifications. Briefly, the yeast culture was harvested at OD 6.0–7.0 and lysed by disruption with glass beads in buffer A (50 mM HEPES (pH 7.6), 400 mM potassium acetate, 1 mM EDTA, 5% glycerol, 5 mM beta-mercaptoethanol, 1 mM benzamidine, 100 μM leupeptin, 10 μM pepstatin A, and 1 mM PMSF). The cell extract was stirred with 100 mM ammonium sulfate and 0.2 % PEI for 1 h, centrifuged, and the supernatant was loaded onto an IgG column. TFIIH was extensively washed with buffer A plus 400 mM ammonium sulfate to dissociate TFIIK, and then washed with buffer B (50 mM

HEPES (pH 7.6), 300 mM potassium acetate, 1 mM EDTA, 5% glycerol, 2 mM DTT, 1 mM benzamidine, 100 μM leupeptin, and 10 μM pepstatin A). After incubation with TEV protease for 15 hrs at 4 °C, TFIIH was eluted with buffer B, and was further purified on Superose 6 (GE Healthcare) in buffer containing 20 mM HEPES (pH 7.6), 300 mM potassium acetate, 5% glycerol, 5 mM DTT.

Reconstitution of PIC, and PIC-∆TFIIK was performed as before (22). Briefly, a promoter

DNA fragment HIS4(–81/+1) (0.5 nmol) was combined with TFIIA (0.75 nmol), TFIIB (0.75 nmol), TBP (0.5 nmol), TFIIE (0.7 nmol), and 10-subunit TFIIH (0.3 nmol) (or equimolar amounts of TFIIH lacking TFIIK) in buffer (500) (20 mM HEPES (pH7.6), 5 mM DTT, 2 mM magnesium acetate, and 5% glycerol, with the mM concentration of potassium acetate in parentheses), and dialyzed in steps of buffer (300), buffer (220), buffer (150) for at least 4h at each step and then combined with pol II-TFIIF (0.25 nmol) and excess TFIIS (0.9 nmol). The mixture was further dialyzed into buffer (100) and buffer (40) before loading on a 10–40%(v/v) glycerol gradient in buffer (30). After centrifugation at 36,000 rpm for 11 hours in a Beckman SW60 rotor, the gradient was fractionated using a PGF Piston Gradient Fractionator (BioComp). For cryo-EM samples, PICs were fixed through glycerol gradients containing a gradient of glutaraldehyde from 0 to 0.125% (30). Aliquots of peak fractions (0.3–0.7 mg/ml) were flash frozen in liquid nitrogen and stored until use at –80°C.

Specimen preparation and electron microscopy 20 µl of glutaraldehyde-fixed sample (0.3–0.7 mg/ml) was dialyzed to a buffer (20 mM HEPES (pH7.6), 5 mM DTT, 2 mM magnesium acetate, and 30–40 mM potassium acetate) for 2-3 hours to remove glycerol using a Slide-A-Lyzer MINI Dialysis Unit (20K MWCO) (Thermo Scientific). After an electron microscopic grid (Quantifoil R2/4 or R2/1) was glow-discharged for 15 sec using a Harrick Plasma Cleaner (PDC-32G), 3–4 μl were transferred to the grid and flash frozen in liquid ethane with a Vitrobot (FEI) at 100% humidity and 22°C (blot time 3–4sec; waiting 10sec; force 9). Images were recorded at a magnification of 81,000 x on a 4k x 4k CCD camera (Gatan) in a Tecnai F20 electron microscope (FEI) equipped with a field-emission gun operated at 200 kV acceleration voltage using low-dose conditions (10–15 e– /Å2 exposures) and a range of defocus values (from –1.5 to –3.5 μm).

Ab initio reconstruction The defocus and astigmatism parameters of the micrographs were determined with CTFFIND (46). The micrographs were corrected for the contrast transfer function (CTF) using a binary phase flipping algorithm available in SPIDER (47). 250,000 PIC particles were windowed semi-automatically using the EMAN program Boxer (48). Image clustering, initial model generation from class averages and refinement of the orientations of the individual particle images were done in SIMPLE (24). Heterogeneity analysis was necessary to extend the resolution beyond 30 Å but the conformational heterogeneity can and will in the future be modeled better with improved methods. 35,000 images (~15 % of the total data set) contributed to the state of PIC that showed the best resolved RNA polymerase II structure.

Refinement Spectrally self-adaptive refinement, which in every cycle updates an automatically determined low-pass limit for each image individually (49), was applied to improve the resolution of the

maps. The low-pass limits are indicative of data and reconstruction quality. Reconstructions that allow for visualization of secondary structure elements produce low-pass limits below 10 Å for a significant fraction of the images. The average resolution limits for the converged reconstructions of the different PIC data sets were around 30 Å and the best resolution limits reached 16 Å.

Determination of the number of states In order to determine the suitable number of state groups to partition the data into, the distributions of common line correlation were analyzed statistically. Two coexisting conformational states give rise to a significant correlation gain when optimizing over two state groups instead of one. There will always be a gain in correlation when the number of parameters of the model is increased—the question is if the distributions of correlations differ significantly. Comparing the 1-state correlation distribution with the 2-states distribution for a binary heterogeneity gives a high probability that the two distributions represent samples taken from different probability density functions (pdfs), whereas comparing the 2-states distribution with the 3-states distribution gives a low probability that the two distributions represent samples taken from the same pdf. We applied a Kolmogorov-Smirnov statistical test to compare the 1 vs. 2, 2 vs. 3, and 3 vs. 4 correlation distributions for all three PIC data sets. The probability that groups 1 and 2 represented samples from the same pdf was lower than 0.1 for all three data sets. The probability that distributions 2 and 3 and distributions 3 and 4 represented samples from the same pdf was higher than 0.7 for all three data sets. We concluded that PIC exists in two predominant conformation states. Assessment of heterogeneity analysis We wanted to additionally assess how well the heterogeneity had been resolved after refinement. Comparing class averages obtained with a model-based refinement procedure with re-projections of the volumes is not meaningful—data put in the 3D Fourier transform and data extracted from the same place will always agree perfectly. Instead, the data were clustered in a reference-free manner (24), class averages were aligned to the refined volumes, and the volumes were re-projected in the determined orientations. This approach is free from model bias, but the agreement between re-projections and reference-free class averages is not expected to be perfect due to two factors. First, classification errors are increased with the reference-free approach. Second, averaging over views with similar but different projection direction degrades the resolution of the class averages. The good agreement between reference-free class averages and re-projections confirms that the major PIC heterogeneity was resolved (Fig. S4). The class average alignment also confirmed that most projection directions were covered—a requirement for accurate 3D reconstruction (Fig. S5).

Model building The structure of pol II–TFIIBN(residues 17–87)(12) modeled with the Rpb4/7 subunits (50) was fitted into the EM density. TFIIB and DNA could be identified by docking a crystallographic model of a “minimal” PIC (14) (pol II-TBP-TFIIB-DNA complex). The promoter DNA was extended in both directions with canonical straight B-DNA duplex. To fit the downstream DNA into the cryo-EM density, the DNA between –53 and –51 was bent about 15° perpendicular to the projection of the maximum bend. When viewed in the projection of maximum bend, the bending angle between the cylindrical axes of incoming and outgoing B-form double helices is

approximately 75° as in the original crystal structure (15) rather than 90° in the previous minimal PIC model(14). TFIIB-TBP–DNA interactions are essentially unchanged compared with those observed in the crystal structure (15).

Cross-linking and digestion The isolated PIC (0.35 mg/ml) was cross-linked by 2.25 mM BS3 cross-linker (Bis (sulfosuccinimidyl) suberate (572.43g/mol), Thermo Fisher Scientific) in 100 µl buffer A (20 mM HEPES (pH7.5), 80 mM potassium acetate, 2 mM magnesium acetate, 5 mM DTT, and 15% glycerol) for 2 hrs on ice. The reaction was quenched by adding 2.9 µl of 1M ammonium bicarbonate for 30 min on ice. The cross-linked complex was precipitated by acetone at –80ºC, and centrifuged at 10000 rpm, 4°C for 10 minutes. The supernatant was discarded and the precipitated protein dried in a SpeedVac. The protein was reconstituted in 15 µL 8M urea, 50 mM ammonium bicarbonate, pH 8.0 and 20 µL of 0.25% protease max (Promega, Madison, WI) in 50 mM ammonium bicarbonate pH 8.0. The sample was reduced with 50 mM DTT at 55°C for 30 minutes and then alkylated with 100 mM propionamide for 30 minutes at room temperature. Digestion volume was adjusted to keep the urea concentration to less than 1 M, and trypsin was added at a 1:50 enzyme to protein ratio. Digestion occurred overnight at 37°C after which the reaction was quenched by the addition of 10 µL 50% formic acid water. The peptides were further purified using microspin columns (The Nest Group, inc.) and the eluted peptides dried in a speed vac.

The peptide digest was fractionated by size exclusion chromatography (SEC) using a previously described method (51). Briefly, the dried digest was resuspended with 20 ul of SEC mobile phase (water/acetonitrile/trifluoroacetic acid, 70:30:0.1) and loaded onto a Superdex Peptide PC 3.2/30 column (GE Healthcare). Resulting fractions were dried to completion. Cross-linked peptides were eventually identified in the 1.0–1.8 mL range of the elution volume.

Mass spectrometry Peptides were loaded onto a self-packed C18 analytical reversed phase column (100 uM ID x 12 cm length) at a flow rate of 300 nL/min following a linear gradient going from 2% acetonitrile in 0.585% acetic acid to 40% acetonitrile in 0.585% formic acid in 80 minutes. The hybrid Orbitrap mass spectrometer was used to detect both the peptides and their fragment ions at high resolution (high-high). A charge state of 3+ and higher was used to trigger the MS/MS analysis followed by dynamic exclusion of 45 second with a 10 ppm exclusion mass window. The data dependent settings were such that HCD was performed on the top 10 most intense precursor ions meeting the predefined qualities, and the repeat count was 1. The tune file was such that 2 microscans were used.

Analysis of the data relied on In-silico trypsin digestion of the thirty two sequences of PIC to give a list of all possible digest peptides with the following parameters: (a) up to four missed cleavages; (b) fixed modification to cystines (proponamide); (c) variable modifications to methionines (oxidation), N-termini (acetylation) and lysines (BS3 mono-link reacted with water or ammonia). An all-against-all cross-linking of lysine residues and N-termini from the digest list gave the list of all possible cross-linked peptide pairs from PIC. This cross-link list has nearly half a billion entries.

The raw MS data files were converted into MGF format using MassMatrix (52). The mass of each precursor ion was compared to the list of possible cross-linked peptides. Cross-linked peptides with a total mass that matched the precursor ion within a tolerance of 6 ppm were

tagged as possible assignment candidates and analysed for MS/MS fit. The expected b- and y-series from each candidate were compared to the measured MS/MS spectrum with the same 6 ppm mass tolerance. The b1 and y1 fragments of the cross-linked peptides were not used. Two scores were assigned to each candidate to quantify its fit to the MS/MS data. Score 1 was defined as: ‘2’ if both peptides had four or more assigned b- and y-fragments each; ‘1’ if one of the peptides had only three assigned b- and y-fragments, and; ‘0’ otherwise. Score 2 was defined as: {# of matching b,y fragments} / {# of residues in both peptides}

After the MS/MS scores of all the candidates were assessed, the precursor ion was assigned to a specific candidate if three conditions were fulfilled: (a) This candidate had S1 score of ‘1’ and S2 score higher than 0.9, or S1 score of ‘2’ and S2 score higher than 0.6. These thresholds were determined to set a false-positive rate of less than 1.5% (see below). (b) This candidate had the highest S2 score of all the other candidate matches to that precursor ion. If two candidates had the same top S2 score, the entire precursor ion was discarded. (c) The precursor ion cannot be assigned to a linear (non-cross-linked) peptide under similar matching criteria. We note that these conditions are very similar to those used by Chen et al. (25). The resulting list of cross-link assignments was subjected to a final filtering step to remove redundancy of identical lysine pairs that originated from alternative trypsin digestion sites: only the occurrence with highest S2 score was kept.

The automatic assignment of the mass-spectrometry data described above was accompanied by a round of manually-supervised assignment with a different S2 score criterion. To that aim, the precursor ion was assigned to a specific candidate if four conditions were fulfilled: (a) This entry had S1 score of ‘1’ or ‘2’. (b) This entry had S2 score that was at least 40% higher than that of the next-best candidate match to that precursor ion. (c) The precursor ion cannot be assigned to a single (non-cross-linked) peptide under similar matching criteria. (d) Manual inspection of the corresponding MS/MS spectrum showed that all, or nearly all, of the high-intensity peaks were assigned to expected b- and y-fragments.

False-positive rate of identified cross-links The false-positive rate was estimated for each cross-link data set separately following Kalisman et al. (27). The estimation relies on the assumption that the false-positive rate would be characterized by the typical number of identified cross-links when an erroneous mass for the cross-linker is used. To that end, we repeated the analysis of the mass-spectrometry data with errors of –5, –4, –3, –2, –1, 1, 2, 3, 4, and 5 Daltons added in turn to the correct BS3 mass. The results are shown in Figs. S10A–C for the data sets of PIC, PIC-ΔTFIIK, and TFIIH, respectively. For the data set of PIC, we see that about 3 cross-links are identified in a typical decoy analysis when the identification thresholds are: S1 score of ‘1’ and S2 score higher than 0.9, or S1 score of ‘2’ and S2 score higher than 0.6. For the same thresholds and the correct BS3 mass the analysis identifies 212 cross-links. Therefore, the false-positive rate for the automatic assignment of the PIC data is 3 out of 212 or slightly less than 1.5%. A similar false-positive rate was observed for the PIC-ΔTFIIK data when the thresholds were S1 score of ‘1’ and S2 score higher than 0.9, or S1 score of ‘2’ and S2 score higher than 0.7.

Modelling based on XL-MS and cryo-EM: Coarse representation of TFIIE and TFIIH We used a highly coarse representation of TFIIE and TFIIH, where each protein subunit was reduced to either one or two spheres (Fig. 5). This representation encompasses nine protein subunits that cross-link extensively with each other (Fig. S7): Tfa1, Tfa2, Ssl2, Rad3, Tfb1,

Tfb2, Ssl1, Tfb4, and Tfb5. The TFIIK trimer (Kin28, Ccl1, and Tfb3) was ignored in this analysis due to scarcity of cross-links from its subunits to the rest of the PIC. Overall, we partitioned the sequences of the nine subunits into 12 domains that were each treated as a single structural element (a sphere). This partitioning is based on inter- and intra-subunit cross-links, as well as the length of the sequence (Fig. S12):

Ssl2. Represented by two domains segmented at the ‘hinge’ region around residue 537. Parsing is based on the reliable homology model to archaeal XPB (53).

Rad3. Represented by a single domain despite its large size. Extensive intra-subunit cross-links, as well as reliable homology to archaeal XPD (54, 55), indicate a compact globular structure.

Tfb1. Represented by two domains. The first domain comprises the N-terminal and the C-terminal thirds, while the second domain comprises the middle third. This parsing is based on proximity of Lys171 and Lys581 supported by their intra-subunit cross-link as well as inter-subunit cross-links to residue K366 of Tfa1.

Tfb2 and Tfb5. Both subunits are unified to a single domain. Inter-subunit cross-links between Tfb5 and the C-terminal region of Tfb2 are in good agreement with the crystal structure of Tfb2-Tfb5 complex(38). We obtained very few inter- or intra- cross-links in the N-terminal region of Tfb2 (residues 1–262), suggesting a location well separated from the rest of TFIIH or a disordered structure.

Ssl1. Represented by two domains comprising half the sequence each. Lack of long-range internal cross-links and the prevalence of cross-links to other subunits along the sequence suggest that Ssl1 is elongated rather than globular.

Tfb4. Represented by a single domain. Tfa1 and Tfa2. Represented by three domains: (i) the N-terminal region (residues 1–170)

of Tfa2; (ii) the C-terminal region (residues 171–328) of Tfa2 and the N-terminal region (residues 1–240) of Tfa1 (hatched); (iii) The C-terminal region (residues 241–482) of Tfa1. The C-terminal region of Tfa2 and the N-terminal region of Tfa1 cross-link to each other and also to the same residues of Rad3. This putative dimerization was previously reported (17, 56, 57).

Modelling based on XL-MS and cryo-EM: Detailed assignment of the G-lobe We describe here in more detail the modelling process shown in Fig. 5. We chose 12 locations that span the EM density attributed to TFIIE and TFIIH and tried to match them with the 12 coarse-representation spheres of these factors. The locations were chosen by an iterative and greedy algorithm: We took the highest density voxel to be our first location. The second location was chosen as the highest density voxel that was farther than 35Å from the first location. The third location was the highest density voxel that was farther than 35Å from the first and second locations. This repeated iteratively twelve times.

There are twelve factorial possible models that assign the twelve spheres to the twelve locations, and we performed an exhaustive analysis of all of them. To ensure proper protein connectivity we discarded models in which two spheres belonging to the same protein were farther than 45Å apart, and were left with 563,903 models. We next evaluate the fit of each model to the cross-link data by two measures that are defined pair-wise on cross-linked spheres, i.e. pairs of spheres whose corresponding sequence segments are cross-linked in either the PIC or PIC-∆TFIIK data sets. The 'serious violation' measure tallies the pairs of sphere that are cross-linked, but separated by more than 65Å. The 'violation distance' is a softer measure that sums the excess distances over 40Å between cross-linked spheres. If a pair of spheres was cross-

linked by several cross-links, only one cross-link instance was considered. This was done to avoid bias toward specific pairs of spheres. Distances corresponding to cross-links between the twelve locations and subunits outside TFIIE and TFIIH, were measured to the relevant Cα atoms in the docked crystal structures or added homology models (Table S5). The consensus table (Fig. 5D) comprises the models whose sums of violation distances are under 300Å.

Supplementary Text

Segmentation of electron density: location of GTFs and DNA Manual fitting of the pol II structure to the P-lobe was optimized with computational rigid-body refinement. Features of the pol II structure, such as the Rpb4 and Rpb7 subunits protruding from the side and the active center cleft adjacent to the “clamp,” were clearly visible (Fig. 1). The clamp itself was poorly defined in the PIC, indicative of conformational flexibility, consistent with movement of the clamp by more than 20 Å between different crystal forms (10) and as measured by FRET in solution (58). A difference map calculated between the pol II structure and the P-lobe revealed one extra region of density in the P-lobe, adjacent to the Rpb4/7 subunits (Fig. S3). The extra density may be due to the C-terminal domain (CTD) of Rpb1, by comparison with an X-ray structure of S. pombe pol II (59). The main chain of Rpb1 terminates in the X-ray structure at the beginning of the CTD, due to motion or disorder, and the extra density in the P-lobe occurs at this location. The G-lobes appeared similar in states 1 and 2 of the PIC (correlation coefficient better than 0.8), with a rotation of the G-lobe relative to the P-lobe between states (data not shown).

The EM maps of the core PIC also contained density attributable to TBP, TFIIA, TFIIB, and promoter DNA (Fig. 3). Density for TBP, TFIIB and DNA could be identified by docking a crystallographic model of a “minimal” PIC (pol II-TBP-TFIIB-DNA complex), derived from X-ray structures of pol II-TFIIB and of C-terminal fragments of TBP and TFIIB bound to a TATA box DNA fragment (13-15). Only two slight deviations from the model were required to fit the DNA to the cryo-EM density: a bend of about 75° rather than 90° at the TATA box, and a path leading over the clamp, running parallel to, rather than directly above, the active center cleft. The path of promoter DNA in our PIC structure is similar to that originally proposed on the basis of a pol II – TFIIB cocrystal structure (12). The bend angle is in agreement with the crystal structure of a TBP-TFIIB-DNA complex (15, 60), and with a determination by cryo-EM for a TFIIA-TBP-TFIIB-pol II complex (Azubel in preparation).

Density for about 80 Å of DNA downstream of the TATA box, corresponding to about 27 bp, was observed (Fig. 3A, B). The DNA density contacted G-lobe density, 10–20 bp downstream of the TATA box (interacting with TFIIE), and merged with Ssl2 densities at the downstream end (Fig. 3A, B). Because roughly cylindrical density for DNA could be seen between the upstream and downstream points of contact, it was essentially free in this region. Although no longer separately visible beyond the point of contact with Ssl2, DNA on a straight path would extend past TFIIK another 10 bp downstream to the end of the promoter fragment. Consistent with such DNA-protein interaction, a barrier to digestion by exonuclease III from the downstream end of the DNA is located at positions –9 and –14 (Fig. S1C) (22).

Cross-linking and mass spectrometry (XL-MS): locations of TFIIA, -B, -E, and -F There were nine cross-links of TFIIF to the Rpb2 subunit of pol II, one cross-link to the Rpb1 subunit, and thirteen cross-links to other GTFs (Fig. 4B and Fig. S7). Most of the cross-links involved the Tfg1-Tfg2 dimerization and the Tfg2 winged-helix (WH) domains of TFIIF. The spatial restraints provided by these cross-links were sufficient to place these domains in the EM map (Fig. 3B). The “flexible arm” of Tfg1 and the “insertion loop” of Tfg2 in the dimerization domain (33) formed a pattern of crosslinks with residues in the Rpb2 subunit of pol II (K87, K344, K358, K426) that placed the dimerization domain adjacent to the Rpb2 “protrusion” and “jaw-lobe” domains (Fig. 4C). The resulting model is in excellent agreement with previous FeBABE cleavage mapping of protein-promoter DNA interaction in complexes formed in yeast nuclear extract (34): the flexible arm is located near the active site of the enzyme, whereas the insertion loop is in proximity to downstream DNA. The location of the insertion loop is supported by cross-links to the C-terminal region of Ssl2, which penetrates the downstream end of the pol II cleft, as shown by cross-linking to Rpb1 residues K1217, 1246, and 1262 (Fig. 4A-C).

The WH domain of Tfg2 formed six cross-links to the Tfa2 subunit of TFIIE, one to the Toa2 subunit of TFIIA (K119) and one to TFIIB (K199), but no cross-links to pol II (Fig. 4B). We generated a homology model of this domain bound to double-stranded DNA by superimposing its NMR structure (61) onto the X-ray structure of FoxO1 bound to DNA (62). We then moved this model along the path of DNA in the PIC and found that only one location satisfied the cross-links to TFIIA and TFIIB without a steric clash (Table S4). This location, at DNA position -51, is juxtaposed to a region of EM density large enough to accommodate TFIIE, thereby satisfying also the cross-links to Tfa2. The density for the WH domain was not resolved from density attributed to TFIIE and DNA (Fig. 3A and B), and was adjacent to but distinct from the globular density assigned to TFIIF.

Comparison of PIC structure from this study with that of Nogales and co-workers While this manuscript was in preparation, Nogales and coworkers reported EM structures of human PICs (41). The Nogales structures were produced by alignment of EM images to a structure of pol II as a search model, so they unavoidably include the pol II structure. Beyond pol II and two GTF polypeptides TBP and TFIIB, there is little resemblance between the Nogales structures and ours (Figs. 6, S13). The major differences between the two structures are as follows:

1. The Nogales structures have no G-lobe (Fig. 6A). The maps contained very little density for TFIIE (30 per cent of expected) and TFIIF (20 per cent) and no density but only a molecular envelope for TFIIH (from a negatively stained rather than a cryo specimen). Density for TBP, TFIIB, TFIIE, and TFIIF was associated with pol II rather than TFIIH. The separation of TFIIE from TFIIH is unlikely, in view of the evidence for strong interaction (Kd ~ 3 nM) between these two factors (39, 63). Our map contained nearly full density for TFIIE (100 per cent), TFIIF (65 per cent), and TFIIH (60 per cent, less than complete due largely to weak density for TFIIK). The association of all the GTFs in the G-lobe accords with their isolation as a stable complex (22) and includes an extensive interaction surface between TFIIE and TFIIH.

2. Even within the pol II-TBP-TFIIA-TFIIB-promoter region of the Nogales structures, with partial density for TFIIF, there are notable differences from our PIC structure. The locations of the proteins appear similar, but the orientations are different, by 20° for TBP-TFIIA-TFIIB with

respect to pol II, by about 90° for the dimerization domain of TFIIF with respect to pol II, and by about 90° for the winged helix domain of Tfg2 (TFIIF) with respect to pol II. These differences are likely due to the low occupancy of TFIIE and the lack of TFIIH in the Nogales structures.

3. The Nogales structure of the PIC including TFIIH is inconsistent with the majority of the intermolecular cross-links involving TFIIE, TFIIF, and TFIIH that we observed. In the case of TFIIH, the human counterparts of yeast Ssl2 and Rad3 were placed at arbitrary positions in the molecular envelope (different by 40 Å and 35 Å from our structure). The location of the remaining volume of TFIIH is about 90 Å from the location in our structure (Fig. 6A-B). Regardless of the positions of the individual core TFIIH subunits within this volume, their placement at this distance would result in serious violations of most of the cross-linking results.

4. A Nogales PIC lacking TFIIH could be reconstructed from cryo-EM images to 11 Å resolution, by straightforward processing of cryo-EM images, because it revealed only limited protein density beyond pol II (for reasons explained below) and was therefore conformationally rigid. The complete PIC that we reconstructed, which revealed almost the entire density for all 31 proteins, was flexible, and therefore conformationally heterogeneous. New methods had to be developed for image processing, and a resolution of 16 Å was obtained.

5. DNA is associated with pol II in the Nogales structures and follows a path inclined downwards from TFIIB, entering the pol II cleft and interacting with Rpb5 (Fig. 6C). In contrast, DNA does not contact pol II at all in our structure, but instead forms part of the G-lobe (Figs. 6B,D); it follows a path approximately parallel to the pol II cleft, suspended above the pol II surface (Fig. S14). In our structure, it is the C-terminal region of Ssl2 rather than DNA that penetrates the pol II cleft in the vicinity of Rpb5.

DNA transactions are fundamental to the mechanism of the PIC, so differences in the DNA path and in the positions of DNA-associated proteins between the between the Nogales structures and ours cannot be due to differences in the source of proteins or the promoter used. Rather, the discrepancy arises, in large part, from the stepwise procedure for PIC assembly employed by Nogales and coworkers. We have found (22), that this approach gives a low yield of complete PIC with poor transcriptional activity, and that the purified complex is heterogeneous, due to the precipitation of TFIIH, and to the loss of TFIIE (also apparent from the raw images of Nogales and coworkers). These deficiencies explain why the Nogales structures show such a small fraction of the total densities for TFIIE and TFIIF, blurred density for TFIIH, and no G-lobe. Another reason is that their reconstruction was performed with the use of a pol II structure as a search model, and could only reveal the parts of the GTFs and DNA that are rigidly bound by pol II. Their reconstructions therefore show principally the pol II-TBP-TFIIA-TFIIB-promoter complex, which is conformationally homogeneous, and could be reconstructed to relatively high resolution. To reveal the rest of the PIC, it is necessary to form a stable, fully stoichiometric complex, which we have accomplished only by the assembly pathway we have described (22). Moreover, reconstruction must be performed in an unbiased manner, rather than with the use of a search model (23); the contribution from pol II is then a result rather than input to the analysis, and the G-lobe is revealed despite flexible linkage to the P-lobe. The flexibility between P- and G-lobes results in conformational heterogeneity, requiring new methods of analysis for refinement beyond 30 Å resolution (24).

Fig. S1 Isolation and characterization of PIC and PIC-ΔTFIIK (A-B) A promoter DNA fragment HIS4(–81/+1) was combined with excess TFIIA, TFIIB, TBP, TFIIE, and 10-subunit TFIIH (A) or TFIIH lacking TFIIK (B) in 0.5 M potassium acetate, and dialyzed to 0.15 M potassium acetate, as previously published (22). The GTF-DNA complex was combined with pol II-TFIIF complex and excess TFIIS, dialyzed to 40 mM potassium acetate, and sedimented on a 10–40% glycerol gradient. The fractions were analyzed by SDS-PAGE and a single peak in the center of the gradient (lanes 5-8) contained PIC (A) or PIC-ΔTFIIK (B). (C) Exonuclease III footprints of the PIC and the partial complexes. The 5’-end of the non-template of HIS4(–93/+109) (22) was labeled with 32P to determine the downstream boundary. Arrows indicate two DNA products that are specific to the intact PIC. The barriers to exonuclease III digestion around –9 and –14 are good agreement with the EM structure (Fig. 3). No barrier was observed when one of the GTFs was omitted.

Fig. S2 Representative images of PIC and PIC-ΔTFIIK Electron micrographs of PIC (A) and PIC-ΔTFIIK(B) embedded in vitreous ice for cryo-EM were recorded with high dose (Imaging conditions: 70 μm objective aperture; defocus 4–5 μm; 25–30 e–/A2 dose). Scale bars = 100 nm.

Fig. S3 A difference map calculated between the P-lobe and the fitted structure of pol II On the left is shown a front view of the PIC (as in Fig. 1A). On the right is shown the crystal structure of S. pombe pol II (Jaw-lobe, cyan; Clamp, yellow; Core, gray ; Shelf, magenta; Rpb4, orange; Rpb7, purple)(59) viewed from the same orientation. A difference map calculated between the P-lobe and the fitted structure of pol II may represent a part of the C-terminal domain (CTD) of Rpb1 (largest residual density in the right panel). The “clamp” and “jaw-lobe” were poorly defined in the PIC, indicative of conformational flexibility(10).

Fig. S4 Comparison between reference-free class averages and re-projections of the refined maps. The good agreement between reference-free class averages and re-projections confirmed that the major PIC heterogeneity had been resolved.

Fig. S5 Scatter plots over the two rotation angles that define a projection direction. Reference-free class averages were aligned to the refined maps. The two states of each PIC samples cover most of the projection directions.

Fig. S6 Fourier Shell Correlation. The resolution of the final density maps was assessed by Fourier Shell Correlation (FSC=0.5) to 16–18 Å. The ‘state 1’-reconstructions correspond to those maps with best agreement between the P-lobe density and the pol II X-ray structure and they are also the best resolved maps (16 Å).

Fig. S7 Schematic representation of the observed cross-links identified by mass-spectrometry. Red dashed lines indicate inter-subunit cross-links derived from PIC, of which 80% were shared with the PIC-ΔTFIIK dataset. Black dashed lines indicate inter-subunit cross-links derived only from PIC-ΔTFIIK. Primary structure is depicted as boxes with a conserved fold in color. Rpb3–Rpb12 of pol II are omitted for clarity.

Fig. S8 Schematic representation of the inter-subunit cross-links of holo-TFIIH identified by mass-spectrometry. Blue dashed lines indicate inter-subunit cross-links derived from holo-TFIIH.

Fig. S9 Observed cross-links between pol II and TFIIB in the PIC. Cross-links between pol II and TFIIB observed in the PIC (blue dashed lines) are in good agreement with the crystal structure of pol II-TFIIB (14). The pol II “clamp” domain (yellow) is apparently highly mobile, as the B-linker region (residues 84–116 of TFIIB) formed six cross-links to the lobe (blue), but not to the clamp that is also within reach of cross-linking distance.

Fig. S10 False-positive rate estimation for the cross-link identification (27). (A) False-positive rate estimation for PIC. The number of identified cross-links is plotted after a deliberate error is added to the true BS3 mass during the analysis. The analysis with the correct BS3 mass identified 212 cross-links (central bar ; Table S1). The median value of identified cross-links in the decoy analyzes is 3 (red dashed line ; range 1-6). The false-positive rate can therefore be estimated to be 3 out of 212 or 1.5%. (B) False-positive rate estimation for PIC-∆TFIIK. The analysis with the correct BS3 mass identified 178 cross-links (central bar; Table S2). The median value of identified cross-links in the decoy analyzes is 3 (red dashed line ; range 0-10). The false-positive rate can therefore be estimated to be 3 out of 212 or 1.5%. (C) False-positive rate estimation for isolated TFIIH. The analysis with the correct BS3 mass identified 112 cross-links (central bar ; Table S3). The median value of identified cross-links in the decoy analyzes is 1 (red dashed line ; range 0-27). The false-positive rate can therefore be estimated to be 1 out of 112 or 1.0%. The number of identified cross-links with a +1Da mass error is 27. This bar was omitted because of its height.

Fig. S11 High resolution fragmentation spectra of cross-linked peptides. (A) Annotated fragmentation MS/MS spectrum that identifies a cross-link between Lys1246 in Rpb1 and Lys827 in Ssl2. The total mass of the cross-linked peptides matches that of the precursor ion (box) to 0.7ppm. The extensive matching of fragments from both peptides to the spectrum provides high confidence in the identification. (B) Annotated fragmentation MS/MS spectrum that identifies a cross-link between Lys537 in Tfg1 and Lys6 in Tfb5. The total mass of the cross-linked peptides matches that of the precursor ion (box) to 1.6ppm. (C) Annotated fragmentation MS/MS spectrum that identifies a cross-link between Lys527 in Tfg1 and Lys55 in TFIIS. The total mass of the cross-linked peptides matches that of the precursor ion (box) to 1.0ppm.

Fig. S12 Structural domains of TFIIE and TFIIH. Cross-links from the PIC and PIC-∆TFIIK datasets are shown as dashed lines. Only internal cross-links that span more than 100 residues are shown. Separated domains are represented in open, shaded, or hatched boxes, as appropriate.

Fig. S13 Bootstrap analysis for domain architecture of TFIIE and TFIIH. We further tested the likelihood of the consensus arrangement (Fig. 5D) by performing a bootstrap analysis. We considered the top 300 arrangements ranked by their Sum of Violation Distance Scores, which varies from 253 to 363 Å. We selected 300 arrangements randomly from these top 300, allowing duplicates to occur. This process makes a different selection for each run and was repeated 1000 times to give the nine different consensus arrangements with their frequencies of occurrence. Three arrangements occurred most often: the top-ranking arrangement occurred in 53% of the runs, the second (ranked 17), which exchanged Tfa1-C and Tfb1-Mid, occurred in 30% of the runs, and the third (ranked 8) occurred in 10% of the runs.

Fig. S14 Promoter DNA in the PIC and in human PIC lacking TFIIH and TFIIE. (A) Front (left) and side (right) views of DNA path in PIC from this work. Pol II is shown

in a surface representation. Densities for TFIIF, TFIIE, and TFIIH are omitted for clarity. The structures of the TBP(green)-TFIIBc(red)-TATA box complex (15) modeled with TFIIA (cyan) (32), are fitted into the EM density as done previously (14). Only two slight deviations from the previous model were required: a bend of about 75° rather than 90° at the TATA box, and a path of the DNA leading over the clamp, adjacent rather than directly above the pol II cleft. The C-terminal region of Ssl2 formed cross-links to K1217, K1246, and K1262 (colored in orange) of the Rpb1 lobe inside the cleft. (B) DNA path in human PIC lacking TFIIH and TFIIE (41) viewed in the same orientations as in (A). The structure of the TBP-TFIIBc-TATA box complex was rotated by 20° compared to (A). The downstream DNA path is partially inside the cleft, and the further downstream it is in contact with Rpb5.

Table S1.

Non-redundant compilation of all cross-links identified for the closed form of PIC. Cross-links with high MS/MS fragmentation scores were assigned automatically. Additional cross-links with slightly lower MS/MS scores were assigned following manual inspection of the MS/MS spectra. The estimated false-positive rate for the cross-links that were assigned automatically is lower than 1.5% (or about 3 out of 212; see Fig. S10).

Score 1 is: '2' for cross-links where each peptide has at least four observed fragments from the b- and y-series; '1' for cross-links where one of the peptides has only three observed fragments from the b- and y-series ; '0' otherwise.

Score 2 is defined as: {# of matching b,y fragments} / {# of residues in both peptides}

Distances refer to the Cα-Cα distance between the two linked lysine residues in cases where a homology model is available.

Mox - oxidized methionine.

acetyl - acetylated N-terminal.

K+ - lysine residue with a BS3 mono-link reacted with water.

K++ - lysine residue with a BS3 mono-link reacted with ammonia.

Table S2. Non-redundant compilation of all cross-links identified for the closed form of PIC-∆TFIIK. The estimated false-positive rate for the cross-links that were assigned automatically is 1.5% (or about 3 out of 178 ; see Fig. S10).

Table S3. Non-redundant compilation of all cross-links identified for TFIIH. The estimated false-positive rate for the cross-links that were assigned automatically is 1.0% (or about 1 out of 112 ; see Fig. S10).

Table S4. Determining the position of the WH domain of Tfg2. The interaction of the WH domain of Tfg2 with double stranded DNA was modeled based on homology (62). This model was moved along the template strand two base-pairs at a time. At each position the fit to the cross-link data was assessed. Positions that resulted in severe steric clash between the domain and other parts of PIC are marked. Only a single position on the DNA satisfied both cross-links without a clash (row marked in green).

Table S5. Distances measured between subunits/domains in the best-fitting model for TFIIE and TFIIH (Fig. 5A). The distances are grouped based on whether they are internal to TFIIE/H (i.e. they occur between two of the twelve locations in the electron density), or external to TFIIE/H (i.e. they occur between a location in the electron density and a lysine residue in the model of ‘minimal-PIC’+TFIIF). The number of different cross-links occurring for each domain-domain interaction is listed. The model suffered only two cross-link violations, one of which, a distance of 65Å between TFIIE subunit Tfa1 K284 and TFIIH subunit Ssl1 K201, could be resolved if the locations of the Tfa1 and Ssl1 spheres were moved closer to each other; the same correction also reduced other, lesser, cross-link violation distances, such as those between the N-terminal part of Ssl1 and TFIIH subunit Tfb2. A second violation of 88Å for a cross-link between Rpb1 K212 and TFIIE subunit Tfa2 K277 may reflect the conformational flexibility of the clamp.

Movie S1

EM density of the PIC and low-pass filtered (20 Å) 12-subunit pol II (45) are viewed from the same orientations.

Movie S2 EM density of the PIC. The structures of the TBP(green)-TFIIBc(red)-TATA box complex (15) modeled with TFIIA (cyan) (32), are fitted into the EM density as done previously (14). Only two slight deviations from the previous model were required: a bend of about 75° rather than 90° at the TATA box, and a path of the DNA leading over the clamp, adjacent rather than directly above the pol II cleft.

Movie S3

PIC showing EM densities attributed to TFIIE (light purple) and TFIIH (core TFIIH, gray; Ssl2, orange; TFIIK, light yellow). Labeled spheres are in locations of TFIIE, core TFIIH, and Ssl2 subunits that best fit the XL-MS data. Other elements of the PIC are represented as in Fig. 5A. Protein Data Bank file of the PIC model (file name: Murakami_et_al_PIC_model.pdb) The model is in register with the deposited electron density of the full PIC (EMDataBank accession code: EMD-2394). The chains in the model describe the following subunits: Chain A – Rpb1 Chain B – Rpb2 Chain C – Rpb3 Chain D – Rpb4 Chain E – Rpb5 Chain F – Rpb6 Chain G – Rpb7 Chain H – Rpb8 Chain I – Rpb9

Chain J – Rpb10 Chain K – Rpb11 Chain L – Rpb12 Chain M – Toa1 Chain O – Toa2 Chain P – TFIIB Chain Q – TBP Chain R – Tfg1 (only the domain associated with the Tfg1/Tfg2 dimerization is modelled; Fig. 4) Chain S – Tfg2 (only the WH domain and the domain associated with the Tfg1/Tfg2 dimerization are modelled; Fig. 4) Chain N – Non-template strand of DNA. Chain T – Template strand of DNA. Chain Z – Coarse grained model of TFIIE and TFIIH (Fig. 5; Table S5). The atoms in chain Z are used only as markers for the coordinates of the coarse-grained spheres in Figure 5. The assignment of subunits in the best fitting model (Fig. 5A) and the position numbers in Fig. 5D correspond to the following atoms in chain Z: Residue 5 – Tfa2-N terminal half; position 1 in Figure 5D Residue 10 – Tfa2-C terminal half and Tfa1-N terminal half ; position 2 in Figure 5D Residue 15 – Tfa1-C terminal half ; position 3 in Figure 5D Residue 20 – Rad3 ; position 4 in Figure 5D Residue 25 – Tfb1-N and C terminal thirds ; position 5 in Figure 5D Residue 30 – Tfb1-middle third ; position 6 in Figure 5D Residue 35 – Tfb4 ; position 7 in Figure 5D Residue 40 – Ssl1-N terminal half ; position 8 in Figure 5D Residue 45 – Ssl1-C terminal half ; position 9 in Figure 5D Residue 50 – Tfb2 and Tfb5 ; position 10 in Figure 5D Residue 55 – Ssl2-N terminal half ; position 11 in Figure 5D Residue 60 – Ssl2-C terminal half ; position 12 in Figure 5D In addition, the following atoms in chain Z describe positions external to TFIIE and TFIIH that were cross-linked to subunits in those factors: Residue 1000 – Rpb1-Lys1217 Residue 1005 – Rpb1-Lys1246 Residue 1010 – Rpb1-Lys1262 Residue 1015 – Rpb1-Lys212 Residue 1020 – Tfg1-C terminal Residue 1025 – Tfg2-Insertion loop in dimerization domain Residue 1030 – Tfg2-WH domain Residue 1035 – TBP-Lys151

Table  S4.    The  fit  to  data  of  a  model  of  the  WH  domain  of  Tfg2  as  it  is  moved  along  the  DNA  path.

DNA  Position Tfg2  K286  to  TFIIB  K199 Tfg2  K286  to  Toa2  K119 Steric  Clash-­‐77 64 63-­‐75 59 54-­‐73 51 44-­‐71 41 40-­‐69 35 41 Yes-­‐67 33 37 Yes-­‐65 29 28 Yes-­‐63 20 22 Yes-­‐61 11 32 Yes-­‐59 18 40 Yes-­‐57 28 41 Yes-­‐55 34 34 Yes-­‐53 31 26-­‐51 21 20-­‐49 14 29-­‐47 23 39 Yes-­‐45 33 43-­‐43 38 42

Cross-­‐link  Distance

Domain  1 Domain  2 #  of  cross-­‐links Distance  [Å] Main  Dataset

Within  TFIIE  and  TFIIH:Tfa1-­‐C Ssl1-­‐N 2 65.3 PIC-­‐ΔTFIIKTfa1-­‐C Tfb4 1 62.7 PICTfa1-­‐N/Tfa2-­‐C Rad3 8 60.8 PIC-­‐ΔTFIIKSsl1-­‐N Tfb4 1 58.1 PICTfb2/Tfb5 Ssl1-­‐N 2 52.4 PICTfa1-­‐C Tfb2/Tfb5 1 52.0 PICTfb1-­‐Mid Ssl1-­‐N 1 46.3 PICRad3 Tfb1-­‐Mid 1 45.1 PICTfa1-­‐N/Tfa2-­‐C Tfb1-­‐Mid 1 43.8 PIC-­‐ΔTFIIKRad3 Ssl1-­‐N 1 41.8 PICTfa1-­‐C Tfb1-­‐N/C 2 41.0 PICTfb1-­‐Mid Ssl1-­‐C 1 37.4 PICSsl1-­‐C Tfb4 4 34.8 PICTfb1-­‐N/C Tfb4 1 31.5 PICTfb2/Tfb5 Tfb4 2 29.2 PIC

External  to  TFIIE  and  TFIIH:Ssl2-­‐C Rpb1-­‐Lys1217 1 63.8 PICSsl2-­‐C Rpb1-­‐Lys1246 1 51.3 PICSsl2-­‐C Rpb1-­‐Lys1262 1 56.6 PICSsl2-­‐C Tfg1/Tfg2-­‐dimerization 2 39.3 PICTfa1-­‐N/Tfa2-­‐C Rpb1-­‐Lys212 1 88.2 PICTfa1-­‐N/Tfa2-­‐C Tfg2-­‐WH 1 52.4 PICTfa2-­‐N TBP-­‐Lys151 1 27.6 PICTfa2-­‐N Tfg2-­‐WH 5 25.2 PICTfb2/Tfb5 Tfg1-­‐Cterm 1 50.9 PIC

Main  Dataset

References and Notes 1. R. C. Conaway, J. W. Conaway, General initiation factors for RNA polymerase II. Annu. Rev.

Biochem. 62, 161–190 (1993). doi:10.1146/annurev.bi.62.070193.001113 Medline

2. R. D. Kornberg, The molecular basis of eukaryotic transcription. Proc. Natl. Acad. Sci. U.S.A. 104, 12955–12961 (2007). doi:10.1073/pnas.0704138104 Medline

3. R. D. Kornberg, Mediator and the mechanism of transcriptional activation. Trends Biochem. Sci. 30, 235–239 (2005). doi:10.1016/j.tibs.2005.03.011 Medline

4. S. K. Burley, R. G. Roeder, Biochemistry and structural biology of transcription factor IID (TFIID). Annu. Rev. Biochem. 65, 769–799 (1996). doi:10.1146/annurev.bi.65.070196.004005 Medline

5. S. Buratowski, S. Hahn, L. Guarente, P. A. Sharp, Five intermediate complexes in transcription initiation by RNA polymerase II. Cell 56, 549–561 (1989). doi:10.1016/0092-8674(89)90578-3 Medline

6. S. N. Guzder, P. Sung, V. Bailly, L. Prakash, S. Prakash, RAD25 is a DNA helicase required for DNA repair and RNA polymerase II transcription. Nature 369, 578–581 (1994). doi:10.1038/369578a0 Medline

7. W. J. Feaver, O. Gileadi, Y. Li, R. D. Kornberg, CTD kinase associated with yeast RNA polymerase II initiation factor b. Cell 67, 1223–1230 (1991). doi:10.1016/0092-8674(91)90298-D Medline

8. Y. J. Kim, S. Björklund, Y. Li, M. H. Sayre, R. D. Kornberg, A multiprotein mediator of transcriptional activation and its interaction with the C-terminal repeat domain of RNA polymerase II. Cell 77, 599–608 (1994). doi:10.1016/0092-8674(94)90221-6 Medline

9. P. Cramer, D. A. Bushnell, J. Fu, A. L. Gnatt, B. Maier-Davis, N. E. Thompson, R. R. Burgess, A. M. Edwards, P. R. David, R. D. Kornberg, Architecture of RNA polymerase II and implications for the transcription mechanism. Science 288, 640–649 (2000). doi:10.1126/science.288.5466.640

10. P. Cramer, D. A. Bushnell, R. D. Kornberg, Structural basis of transcription: RNA polymerase II at 2.8 angstrom resolution. Science 292, 1863–1876 (2001). doi:10.1126/science.1059493

11. A. L. Gnatt, P. Cramer, J. Fu, D. A. Bushnell, R. D. Kornberg, Structural basis of transcription: an RNA polymerase II elongation complex at 3.3 A resolution. Science 292, 1876–1882 (2001). doi:10.1126/science.1059495

12. D. A. Bushnell, K. D. Westover, R. E. Davis, R. D. Kornberg, Structural basis of transcription: an RNA polymerase II-TFIIB cocrystal at 4.5 Angstroms. Science 303, 983–988 (2004). doi:10.1126/science.1090838

13. D. Kostrewa, M. E. Zeller, K. J. Armache, M. Seizl, K. Leike, M. Thomm, P. Cramer, RNA polymerase II-TFIIB structure and mechanism of transcription initiation. Nature 462, 323–330 (2009). doi:10.1038/nature08548 Medline

14. X. Liu, D. A. Bushnell, D. Wang, G. Calero, R. D. Kornberg, Structure of an RNA polymerase II-TFIIB complex and the transcription initiation mechanism. Science 327, 206–209 (2010). doi:10.1126/science.1182015

15. D. B. Nikolov, H. Chen, E. D. Halay, A. A. Usheva, K. Hisatake, D. K. Lee, R. G. Roeder, S. K. Burley, Crystal structure of a TFIIB-TBP-TATA-element ternary complex. Nature 377, 119–128 (1995). doi:10.1038/377119a0 Medline

16. W. H. Chung, J. L. Craighead, W. H. Chang, C. Ezeokonkwo, A. Bareket-Samish, R. D. Kornberg, F. J. Asturias, RNA polymerase II/TFIIF structure and conserved organization of the initiation complex. Mol. Cell 12, 1003–1013 (2003). doi:10.1016/S1097-2765(03)00387-3 Medline

17. A. Jawhari, M. Uhring, S. De Carlo, C. Crucifix, G. Tocchini-Valentini, D. Moras, P. Schultz, A. Poterszman, Structure and oligomeric state of human transcription factor TFIIE. EMBO Rep. 7, 500–505 (2006). Medline

18. W. H. Chang, R. D. Kornberg, Electron crystal structure of the transcription factor and DNA repair complex, core TFIIH. Cell 102, 609–613 (2000). doi:10.1016/S0092-8674(00)00083-0 Medline

19. P. Schultz, S. Fribourg, A. Poterszman, V. Mallouh, D. Moras, J. M. Egly, Molecular structure of human TFIIH. Cell 102, 599–607 (2000). doi:10.1016/S0092-8674(00)00082-9 Medline

20. B. J. Gibbons, E. J. Brignole, M. Azubel, K. Murakami, N. R. Voss, D. A. Bushnell, F. J. Asturias, R. D. Kornberg, Subunit architecture of general transcription factor TFIIH. Proc. Natl. Acad. Sci. U.S.A. 109, 1949–1954 (2012). doi:10.1073/pnas.1105266109 Medline

21. K. Murakami, B. J. Gibbons, R. E. Davis, S. Nagai, X. Liu, P. J. Robinson, T. Wu, C. D. Kaplan, R. D. Kornberg, Tfb6, a previously unidentified subunit of the general transcription factor TFIIH, facilitates dissociation of Ssl2 helicase after transcription initiation. Proc. Natl. Acad. Sci. U.S.A. 109, 4816–4821 (2012). doi:10.1073/pnas.1201448109 Medline

22. K. Murakami, G. Calero, C. R. Brown, X. Liu, R. E. Davis, H. Boeger, R. D. Kornberg, Formation and fate of a complete 31-protein RNA polymerase II transcription preinitiation complex. J. Biol. Chem. 288, 6325–6332 (2013). doi:10.1074/jbc.M112.433623 Medline

23. D. Elmlund, R. Davis, H. Elmlund, Ab initio structure determination from electron microscopic images of single molecules coexisting in different functional states. Structure 18, 777–786 (2010). doi:10.1016/j.str.2010.06.001 Medline

24. D. Elmlund, H. Elmlund, SIMPLE: Software for ab initio reconstruction of heterogeneous single-particles. J. Struct. Biol. 180, 420–427 (2012). doi:10.1016/j.jsb.2012.07.010 Medline

25. Z. A. Chen, A. Jawhari, L. Fischer, C. Buchen, S. Tahir, T. Kamenski, M. Rasmussen, L. Lariviere, J. C. Bukowski-Wills, M. Nilges, P. Cramer, J. Rappsilber, Architecture of the RNA polymerase II-TFIIF complex revealed by cross-linking and mass spectrometry. EMBO J. 29, 717–726 (2010). doi:10.1038/emboj.2009.401 Medline

26. K. Lasker, F. Förster, S. Bohn, T. Walzthoeni, E. Villa, P. Unverdorben, F. Beck, R. Aebersold, A. Sali, W. Baumeister, Molecular architecture of the 26S proteasome holocomplex determined by an integrative approach. Proc. Natl. Acad. Sci. U.S.A. 109, 1380–1387 (2012). doi:10.1073/pnas.1120559109 Medline

27. N. Kalisman, C. M. Adams, M. Levitt, Subunit order of eukaryotic TRiC/CCT chaperonin by cross-linking, mass spectrometry, and combinatorial homology modeling. Proc. Natl. Acad. Sci. U.S.A. 109, 2884–2889 (2012). doi:10.1073/pnas.1119472109 Medline

28. D. M. Prather, E. Larschan, F. Winston, Evidence that the elongation factor TFIIS plays a role in transcription initiation at GAL1 in Saccharomyces cerevisiae. Mol. Cell. Biol. 25, 2650–2659 (2005). doi:10.1128/MCB.25.7.2650-2659.2005 Medline

29. B. Kim, A. I. Nesvizhskii, P. G. Rani, S. Hahn, R. Aebersold, J. A. Ranish, The transcription elongation factor TFIIS is a component of RNA polymerase II preinitiation complexes. Proc. Natl. Acad. Sci. U.S.A. 104, 16068–16073 (2007). doi:10.1073/pnas.0704573104 Medline

30. B. Kastner, N. Fischer, M. M. Golas, B. Sander, P. Dube, D. Boehringer, K. Hartmuth, J. Deckert, F. Hauer, E. Wolf, H. Uchtenhagen, H. Urlaub, F. Herzog, J. M. Peters, D. Poerschke, R. Lührmann, H. Stark, GraFix: sample preparation for single-particle electron cryomicroscopy. Nat. Methods 5, 53–55 (2008). doi:10.1038/nmeth1139 Medline

31. H. Kettenberger, K. J. Armache, P. Cramer, Complete RNA polymerase II elongation complex structure and its interactions with NTP and TFIIS. Mol. Cell 16, 955–965 (2004). doi:10.1016/j.molcel.2004.11.040 Medline

32. S. Tan, Y. Hunziker, D. F. Sargent, T. J. Richmond, Crystal structure of a yeast TFIIA/TBP/DNA complex. Nature 381, 127–134 (1996). doi:10.1038/381127a0 Medline

33. F. Gaiser, S. Tan, T. J. Richmond, Novel dimerization fold of RAP30/RAP74 in human TFIIF at 1.7 A resolution. J. Mol. Biol. 302, 1119–1127 (2000). doi:10.1006/jmbi.2000.4110 Medline

34. G. Miller, S. Hahn, A DNA-tethered cleavage probe reveals the path for promoter DNA in the yeast preinitiation complex. Nat. Struct. Mol. Biol. 13, 603–610 (2006). doi:10.1038/nsmb1117 Medline

35. T. K. Kim, R. H. Ebright, D. Reinberg, Mechanism of ATP-dependent promoter melting by transcription factor IIH. Science 288, 1418–1421 (2000). doi:10.1126/science.288.5470.1418

36. F. C. Holstege, U. Fiedler, H. T. Timmers, Three transitions in the RNA polymerase II transcription complex during initiation. EMBO J. 16, 7468–7480 (1997). doi:10.1093/emboj/16.24.7468 Medline

37. M. Pal, A. S. Ponticelli, D. S. Luse, The role of the transcription bubble and TFIIB in promoter clearance by RNA polymerase II. Mol. Cell 19, 101–110 (2005). doi:10.1016/j.molcel.2005.05.024 Medline

38. D. E. Kainov, M. Vitorino, J. Cavarelli, A. Poterszman, J. M. Egly, Structural basis for group A trichothiodystrophy. Nat. Struct. Mol. Biol. 15, 980–984 (2008). doi:10.1038/nsmb.1478 Medline

39. M. Okuda, A. Tanaka, M. Satoh, S. Mizuta, M. Takazawa, Y. Ohkuma, Y. Nishimura, Structural insight into the TFIIE-TFIIH interaction: TFIIE and p53 share the binding region on TFIIH. EMBO J. 27, 1161–1171 (2008). doi:10.1038/emboj.2008.47 Medline

40. M. Okuda, Y. Watanabe, H. Okamura, F. Hanaoka, Y. Ohkuma, Y. Nishimura, Structure of the central core domain of TFIIEbeta with a novel double-stranded DNA-binding surface. EMBO J. 19, 1346–1356 (2000). doi:10.1093/emboj/19.6.1346 Medline

41. Y. He, J. Fang, D. J. Taatjes, E. Nogales, Structural visualization of key steps in human transcription initiation. Nature 495, 481–486 (2013). doi:10.1038/nature11991 Medline

42. T. K. Kim, T. Lagrange, Y. H. Wang, J. D. Griffith, D. Reinberg, R. H. Ebright, Trajectory of DNA in the RNA polymerase II transcription preinitiation complex. Proc. Natl. Acad. Sci. U.S.A. 94, 12268–12273 (1997). doi:10.1073/pnas.94.23.12268 Medline

43. D. Forget, M. F. Langelier, C. Thérien, V. Trinh, B. Coulombe, Photo-cross-linking of a purified preinitiation complex reveals central roles for the RNA polymerase II mobile clamp and TFIIE in initiation mechanisms. Mol. Cell. Biol. 24, 1122–1131 (2004). doi:10.1128/MCB.24.3.1122-1131.2004 Medline

44. V. Mekler, E. Kortkhonjia, J. Mukhopadhyay, J. Knight, A. Revyakin, A. N. Kapanidis, W. Niu, Y. W. Ebright, R. Levy, R. H. Ebright, Structural organization of bacterial RNA polymerase holoenzyme and the RNA polymerase-promoter open complex. Cell 108, 599–614 (2002). doi:10.1016/S0092-8674(02)00667-0 Medline

45. D. A. Bushnell, R. D. Kornberg, Complete, 12-subunit RNA polymerase II at 4.1-A resolution: implications for the initiation of transcription. Proc. Natl. Acad. Sci. U.S.A. 100, 6969–6973 (2003). doi:10.1073/pnas.1130601100 Medline

46. J. A. Mindell, N. Grigorieff, Accurate determination of local defocus and specimen tilt in electron microscopy. J. Struct. Biol. 142, 334–347 (2003). doi:10.1016/S1047-8477(03)00069-8 Medline

47. J. Frank, M. Radermacher, P. Penczek, J. Zhu, Y. Li, M. Ladjadj, A. Leith, SPIDER and WEB: processing and visualization of images in 3D electron microscopy and related fields. J. Struct. Biol. 116, 190–199 (1996). doi:10.1006/jsbi.1996.0030 Medline

48. S. J. Ludtke, P. R. Baldwin, W. Chiu, EMAN: semiautomated software for high-resolution single-particle reconstructions. J. Struct. Biol. 128, 82–97 (1999). doi:10.1006/jsbi.1999.4174 Medline

49. D. Elmlund, H. Elmlund, High-resolution single-particle orientation refinement based on spectrally self-adapting common lines. J. Struct. Biol. 167, 83–94 (2009). doi:10.1016/j.jsb.2009.04.009 Medline

50. K. J. Armache, S. Mitterweger, A. Meinhart, P. Cramer, Structures of complete RNA polymerase II and its subcomplex, Rpb4/7. J. Biol. Chem. 280, 7131–7134 (2005). doi:10.1074/jbc.M413038200 Medline

51. A. Leitner et al., Expanding the chemical cross-linking toolbox by the use of multiple proteases and enrichment by size exclusion chromatography. Mol Cell Proteomics 11, M111 014126 (Mar, 2012).

52. H. Xu, M. A. Freitas, MassMatrix: a database search program for rapid characterization of proteins and peptides from tandem mass spectrometry data. Proteomics 9, 1548–1555 (2009). doi:10.1002/pmic.200700322 Medline

53. L. Fan, A. S. Arvai, P. K. Cooper, S. Iwai, F. Hanaoka, J. A. Tainer, Conserved XPB core structure and motifs for DNA unwinding: implications for pathway selection of transcription or excision repair. Mol. Cell 22, 27–37 (2006). doi:10.1016/j.molcel.2006.02.017 Medline

54. L. Fan, J. O. Fuss, Q. J. Cheng, A. S. Arvai, M. Hammel, V. A. Roberts, P. K. Cooper, J. A. Tainer, XPD helicase structures and activities: insights into the cancer and aging phenotypes from XPD mutations. Cell 133, 789–800 (2008). doi:10.1016/j.cell.2008.04.030 Medline

55. H. Liu, J. Rudolf, K. A. Johnson, S. A. McMahon, M. Oke, L. Carter, A. M. McRobbie, S. E. Brown, J. H. Naismith, M. F. White, Structure of the DNA repair helicase XPD. Cell 133, 801–812 (2008). doi:10.1016/j.cell.2008.04.029 Medline

56. T. Okamoto, S. Yamamoto, Y. Watanabe, T. Ohta, F. Hanaoka, R. G. Roeder, Y. Ohkuma, Analysis of the role of TFIIE in transcriptional regulation through structure-function studies of the TFIIEbeta subunit. J. Biol. Chem. 273, 19866–19876 (1998). doi:10.1074/jbc.273.31.19866 Medline

57. S. Grünberg, L. Warfield, S. Hahn, Architecture of the RNA polymerase II preinitiation complex and mechanism of ATP-dependent promoter opening. Nat. Struct. Mol. Biol. 19, 788–796 (2012). doi:10.1038/nsmb.2334 Medline

58. A. Chakraborty, D. Wang, Y. W. Ebright, Y. Korlann, E. Kortkhonjia, T. Kim, S. Chowdhury, S. Wigneshweraraj, H. Irschik, R. Jansen, B. T. Nixon, J. Knight, S. Weiss, R. H. Ebright, Opening and closing of the bacterial RNA polymerase clamp. Science 337, 591–595 (2012). doi:10.1126/science.1218716 Medline

59. H. Spåhr, G. Calero, D. A. Bushnell, R. D. Kornberg, Schizosacharomyces pombe RNA polymerase II at 3.6-A resolution. Proc. Natl. Acad. Sci. U.S.A. 106, 9185–9190 (2009). doi:10.1073/pnas.0903361106 Medline

60. O. Littlefield, Y. Korkhin, P. B. Sigler, The structural basis for the oriented assembly of a TBP/TFB/promoter complex. Proc. Natl. Acad. Sci. U.S.A. 96, 13668–13673 (1999). doi:10.1073/pnas.96.24.13668 Medline

61. C. M. Groft, S. N. Uljon, R. Wang, M. H. Werner, Structural homology between the Rap30 DNA-binding domain and linker histone H5: implications for preinitiation complex assembly. Proc. Natl. Acad. Sci. U.S.A. 95, 9117–9122 (1998). doi:10.1073/pnas.95.16.9117 Medline

62. M. M. Brent, R. Anand, R. Marmorstein, Structural basis for DNA recognition by FoxO1 and its regulation by posttranslational modification. Structure 16, 1407–1416 (2008). doi:10.1016/j.str.2008.06.013 Medline

63. D. A. Bushnell, C. Bamdad, R. D. Kornberg, A minimal set of RNA polymerase II transcription protein interactions. J. Biol. Chem. 271, 20170–20174 (1996). doi:10.1074/jbc.271.33.20170 Medline