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Near-atomic resolution visualization of human transcription promoter opening

Abstract

In eukaryotic transcription initiation, a large multi-subunit pre-initiation complex (PIC) that assembles at the core promoter is required for the opening of the duplex DNA and identification of the start site for transcription by RNA polymerase II. Here we use cryo-electron microscropy (cryo-EM) to determine near-atomic resolution structures of the human PIC in a closed state (engaged with duplex DNA), an open state (engaged with a transcription bubble), and an initially transcribing complex (containing six base pairs of DNA–RNA hybrid). Our studies provide structures for previously uncharacterized components of the PIC, such as TFIIE and TFIIH, and segments of TFIIA, TFIIB and TFIIF. Comparison of the different structures reveals the sequential conformational changes that accompany the transition from each state to the next throughout the transcription initiation process. This analysis illustrates the key role of TFIIB in transcription bubble stabilization and provides strong structural support for a translocase activity of XPB.

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Figure 1: Cryo-EM reconstructions of human PIC in different states during the initiation process.
Figure 2: Examples of near-atomic resolution regions.
Figure 3: Newly visualized structural elements of the human PIC.
Figure 4: Structural transitions during promoter opening.
Figure 5: Nucleic acids rearrangements between stages of transcription.
Figure 6: Possible region of DNA scrunching during initiation.

Accession codes

Primary accessions

Electron Microscopy Data Bank

Protein Data Bank

Data deposits

Cryo-EM density maps have been deposited in the Electron Microscopy Data Bank (EMDB) under accession numbers EMD-8132 (OC), EMD-8133 (ITC), EMD-8134 (ITC(−IIS)), EMD-8135 (CC core), EMD-8136 (OC core), EMD-8137 (ITC core), EMD-8138 (ITC(−IIS) core), EMD-8131 (TFIIH core). Model coordinates have been deposited in the Protein Data Bank (PDB) under accession numbers 5IY6 (CC), 5IY7 (OC), 5IY8 (ITC), 5IY9 (ITC(−IIS)), 5IYA (CC core), 5IYB (OC core), 5IYC (ITC core), 5IYD (ITC(−IIS) core), 5IVW (TFIIH core).

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Acknowledgements

We thank S. Zheng for providing XPB mAb, D. King for providing peptides, P. Grob and T. Houweling for electron microscopy and computer support, respectively; S. Scheres for advice on image processing, S. Kassube for providing TFIIS, and G. Cardone for advice concerning filtering according to local resolution. This work was funded by NIGMS (GM63072 to E.N. and GM110387 to I.I.) and the National Science Foundation (MCB-1149521 to I.I.). Computational resources were provided in part by XSEDE (CHE110042) and the National Energy Research for Scientific Computing Center (DE-AC02-05CH11231). E.N. and R.T are Howard Hughes Medical Institute Investigators.

Author information

Authors and Affiliations

Authors

Contributions

Y.H. designed and carried out the experiments; C.Y. and I.I. performed structural modelling; C.I., J.F. and Y.H. purified GTFs and Pol II; Y.H. and E.N. analysed the data and wrote the paper.

Corresponding authors

Correspondence to Yuan He or Eva Nogales.

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Competing interests

The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Purification and activity of human PIC.

a, SDS–PAGE (4–12% gradient gel followed by silver staining) of purified transcription factors and assembled PIC on a promoter DNA. b, The purified PIC in a was ran on a 10% TBE-urea gel after supplying ribonucleoside triphosphates in a run-off reaction. kDa, kilodaltons.

Extended Data Figure 2 Cryo-EM of the human CC.

a, Representative raw micrograph. b, FSC curve and estimated resolution using the 0.143 criteria following the gold-standard procedure implemented in RELION for both the holo-complex and the PIC core. c, Refinement strategy for the holo-complex (see Methods). The local resolution estimation shows flexibility for TFIIH. Further 3D classification revealed the range of motion of TFIIH within the complex (pink and sky blue densities). Focused refinement on the PIC core (masking out TFIIH) improved alignment accuracy and improved the resolution for the core complex.

Extended Data Figure 3 Cryo-EM of the human OC/ITC/ITC(−IIS) complexes.

a, d, g, Representative raw micrograph. b, e, h, FSC curve and estimated resolution using the 0.143 criteria following the gold-standard procedure implemented in RELION. c, f, i, Refinement strategy for the holo-complex (see Methods). The local resolution estimation shows flexibility for TFIIH. Further 3D classification revealed the range of motion of TFIIH within the complex (pink and blue densities). Notice that the direction and range of motion for these three states is similar. Compared to the CC, the pink densities are approximately the same, but the direction of motion from pink to blue has changed. j, Movement of TFIIH with respect to the core PIC from the CC to OC state.

Extended Data Figure 4 Cryo-EM of the core human OC/ITC/ITC(−IIS) complexes.

a, d, g, Dose-dependent B-factor plot from RELION. b, e, h, FSC curve and estimated resolution using the 0.143 criteria following the gold standard procedure implemented in RELION. c, f, i, Refinement strategy (see Methods). The local resolution estimation and final density map (filtered according to this local resolution) is shown for two different views and at two different thresholds (the higher threshold allows better visualization of the highest resolution features for the more stable elements).

Extended Data Figure 5 Segmented cryo-EM densities and models for different regions of interest within the human core PIC in the CC, OC, ITC and ITC(−IIS) states.

a, TFIIB and its interaction with DNA. b, Extension of TFIIA stabilized by interaction with TBP. c, Trigger loop in Pol II, viewed in an ‘off’ state by comparison with crystallographic structures of yeast Pol II (2NVZ25 in blue and 1Y1V26 in green). d, TFIIE and its interacting partners with the PIC. e, TFIIF and its interacting partners with the PIC.

Extended Data Figure 6 Cryo-EM reconstruction and interpretation of human TFIIH.

a, Refinement strategy (see Methods) for the seven subunit TFIIH core complex (the CAK subcomplex is too flexible to be visualized following averaging). The local resolution estimation for the PIC structure obtained by combining the OC, ITC and ITC(−IIS) data sets shows lower resolution for TFIIH, reflecting its flexible attachment. Focused refinement (using the mask marked by dashed lines) allowed us to improve the resolution for TFIIH. bd, Comparison of human XPB and the ssoRad54 ATPase structures and their interaction with DNA. b, Human XPB–DNA model. N terminus is shown in navy blue, C terminus in pink and DNA in cyan. c, Crystal structure of ssoRad54 ATPase (PDB: 1Z63)33. The N terminus is shown in yellow, the C terminus in green and the DNA in cyan. Whereas the N-terminal domains of both proteins and the DNA can be easily superimposed, the C-terminal domains are in very different position. d, A 123° rotation would be required to superimpose the C-terminal domains around an axis located on the first residues (top, shown in spheres) that connects to the fixed N-terminal domain. The rotation axis and planes are coloured in red. e, Possible location of the TFIIEα–TFIIH/p62 interface. An unassigned density in the region of proximity/contact between TFIIE and TFIIH probably corresponding to the TFIIE–TFIIH interface is marked with boxes for each reconstruction. A tentative orientation of the NMR structure for a short C-terminal segment of TFIIEα bound to the PHD domain of p62 (PDB: 2RNR)94 is proposed in the centre for that flexible density region.

Extended Data Figure 7 Position of the TBP–IIA–IIB module and mobile elements of Pol II.

ac, Comparison between a ‘synthetic’ structure of the CC, generated by superimposing human TBP–TFIIA–DNA (PDB: 1NVP)21, human TBP–TFIIB–DNA (PDB: 2C9B)38, and, most notably, the yeast Pol II–TFIIB (PDB: 4BBR)10 using common elements (green), and the human cryo-EM CC model (coloured). A number of elements are rotated between the two: the TBP-IIA-IIB-DNA subcomplex (a), the Pol II clamp (b), and the Pol II stalk (c). The rotation plane and angle are depicted in red. d, Comparison, shown in two different views, of a recently reported cryo-EM structure of yeast CC (green)13 and the human CC in this study. The structures were aligned using the rigid part of Pol II (that is, excluding the clamp and the stalk). The yeast TBP–TFIIA–TFIIB–DNA module and the mobile regions of Pol II (clamp and stalk) are in different relative positions. Whereas the TBP–TFIIA–TFIIB module and the clamp and stalk element resembles those in the ‘synthetic’ model (ac), the path of the DNA is very different, in that it moves away, rather than towards the Pol II stalk.

Extended Data Figure 8 Comparison of eukaryotic and bacterial initiation complexes around the active site.

a, b, Equivalent close-up views of our ITC(−IIS) model (a) and the crystallographic structure of a bacterial initiation complex (4G7O42) (b). The fork loop 2 is tilted in a very similar manner to that observed in our OC, ITC and ITC(−IIS) structures (see Figs 2b and 5f). Domain 2 within the bacterial σ factor is involved in stabilizing the non-template DNA in a similar manner as the TFIIB linker region in human initiation complex.

Extended Data Figure 9 Comparison of selected regions of the human core PIC structures for the CC, OC, ITC and ITC(−IIS) states.

a, Segmented density and model for TFIIB and Pol II loops critical for stabilization of the transcription bubble. The density for the nucleic acids has been omitted for clarity. b, Interaction of the Pol II clamp head with TFIIE. As the clamp closes down during promoter opening (CC to OC transition), the region of contact with TFIIE changes. c, Interaction of the Pol II clamp head with DNA near the promoter melting site. d, Interaction of RPB5 with DNA. e, Opening of two extra base pairs of DNA in the OC scaffold. EM density and the corresponding model near the initiation bubble upstream fork in the OC (left) and ITC(−IIS) (right) structures. Positions of the duplexed DNA downstream of the BRE were labelled relative to the +1 active site in each structure. The cartoon (bottom) shows the two aligned DNA templates for reference.

Extended Data Table 1 Statistics after refinement of atomic coordinates with Phenix for the core PIC models (OC, ITC, ITC(−IIS) and CC), and scores from MolProbilty structural analysis

Supplementary information

Cryo-EM reconstruction and MDFF model of core ITC

Densities are shown as a semi-transparent surface following a similar colour scheme to that in the main section. A region corresponding to the double-psi beta-barrel domain composing the conserved core of Pol II within Rpb1 and Rpb2 is shown as an example of near-atomic resolution features (as in Fig. 2). Colour scheme for the promoter DNA is shown at the bottom. (MOV 29042 kb)

Cryo-EM reconstruction and MDFF model of core ITC(-IIS)

Densities are shown as a semi-transparent surface following a similar colour scheme to that in the main section. A region corresponding to the active site of Pol II is shown as an example of near-atomic resolution features (as in Fig. 2). Color scheme for the promoter DNA is shown at the bottom. (MOV 28847 kb)

Transitions of nucleic acids through the transcription initiation process

By aligning the three models of holo-PICs (CC, OC, and ITC) using the rigid part of Pol II, sequential states are morphed with a special focus on the nucleic acids regions. (MOV 18290 kb)

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He, Y., Yan, C., Fang, J. et al. Near-atomic resolution visualization of human transcription promoter opening. Nature 533, 359–365 (2016). https://doi.org/10.1038/nature17970

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