Structural basis of RNA polymerase III transcription initiation

Abstract

RNA polymerase (Pol) III transcribes essential non-coding RNAs, including the entire pool of transfer RNAs, the 5S ribosomal RNA and the U6 spliceosomal RNA, and is often deregulated in cancer cells. The initiation of gene transcription by Pol III requires the activity of the transcription factor TFIIIB to form a transcriptionally active Pol III preinitiation complex (PIC). Here we present electron microscopy reconstructions of Pol III PICs at 3.4–4.0 Å and a reconstruction of unbound apo-Pol III at 3.1 Å. TFIIIB fully encircles the DNA and restructures Pol III. In particular, binding of the TFIIIB subunit Bdp1 rearranges the Pol III-specific subunits C37 and C34, thereby promoting DNA opening. The unwound DNA directly contacts both sides of the Pol III cleft. Topologically, the Pol III PIC resembles the Pol II PIC, whereas the Pol I PIC is more divergent. The structures presented unravel the molecular mechanisms underlying the first steps of Pol III transcription and also the general conserved mechanisms of gene transcription initiation.

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Figure 1: Cryo-EM structure of the Saccharomyces cerevisiae RNA Pol III PIC.
Figure 2: Architecture of TFIIIB subunits and upstream DNA assembly.
Figure 3: Promoter opening and template strand loading.
Figure 4: Structural rearrangements upon open complex formation.
Figure 5: The C82–C34–C31 subcomplex is a TFIIF–TFIIE hybrid.

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Acknowledgements

We thank N. Cronin at the Institute of Cancer Research for help with yeast fermentation; C. Richardson for the computing infrastructure; C. Plaschka (MRC-LMB, Cambridge) for advice during data processing; and the staff at beamline M03 of the Diamond Light Source synchrotron (UK) for help with EM data collection (EM15629, EM16599 and EM166601). We acknowledge support and the use of resources of iNEXT, in particular C. Sachse and W. Hagen for EM data collection at EMBL Heidelberg (PID1956 and 2180). G.A.-P. is a recipient of a Marie Sklodowska-Curie Intra-European Fellowship (EU project 655238). E.M. is supported by Cancer Research UK (CR-UK C12209/A16749). A.V. is supported by a Biotechnology and Biological Sciences Research Council (BBSRC) New Investigator Award (BB/K014390/1), a Cancer Research UK Programme Foundation (CR-UK C47547/A21536) and a Wellcome Trust Investigator Award (200818/Z/16/Z).

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Authors

Contributions

G.A.-P. carried out yeast fermentation, Pol III purification, Pol III PIC reconstitution, EM specimen preparation, EM data collection and processing, model building and refinement. E.P.R. carried out EM data collection and processing of the Bdp1Δ(355–372) Pol III PIC and helped with EM data processing and analysis. F.B. carried out cryo-EM sample preparation, screening and sample collection. E.M. helped during initial EM characterization and data collection. A.V. designed and supervised research, analysed the structural data and prepared the manuscript with contributions from all authors.

Corresponding author

Correspondence to Alessandro Vannini.

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The authors declare no competing financial interests.

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Reviewer Information Nature thanks R. Maraia and the other anonymous reviewer(s) for their contribution to the peer review of this work.

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Extended data figures and tables

Extended Data Figure 1 Cryo-EM reconstructions and model fitting.

a, Cryo-EM reconstruction of the OC-PIC (left). Pol III core density is coloured in transparent grey and the TFIIIB, heterodimer, heterotrimer, stalk and DNA are coloured as indicated. Atomic models are represented as ribbon (right). Representative electron microscopy densities of different regions show the detail of the final reconstruction, where amino-acid side-chains are discernible as well as secondary structure features. Modelled DNA nucleotides are represented as indicated in Fig. 1. b, As in a, but for the OC1-POL3 reconstruction. c, As in a, but for the OC2-POL3 reconstruction. d, As in a, but for the oPOL3 reconstruction. e, As in a, but for the cPOL3 reconstruction.

Extended Data Figure 2 Cryo-EM data processing.

a, d, Representative raw micrographs of the Bdp1 wild-type (a) and Bdp1(∆355–372) (d) datasets. b, e, Fifteen representative reference-free 2D class averages of the wild-type Bdp1 (b) and Bdp1(∆355–372) (e) datasets. c, 3D classification of the Bdp1 wild-type data set. The particles were subjected to a hierarchical process, which encompassed several rounds of classification using global masks or masks of specific regions of the complex, as indicated. The number of particles contributing to each class is indicated. The presence of densities corresponding to TFIIIB or the downstream DNA, which guided the classification process, is represented by red or green circles, respectively. f, As in c, but for the 3D classification of the Bdp1(∆355–372) dataset.

Extended Data Figure 3 Resolution of cryo-EM reconstructions.

a, Left, Fourier shell correlation plot of the OC-PIC reconstruction with the estimated resolution at the gold-standard FSC (FSC = 0.143). Middle, lateral view of the orientation distribution sphere of the particles that contributed to the OC-PIC reconstruction. The heights of the surface bars indicate the relative number of particles in a given orientation. Right, resolution estimation represented by a lateral view and central slice of the OC-PIC cryo-EM map The map is coloured according to the local resolution, as indicated in the scale bar. Local resolution was calculated with ResMap 1.1.456 as implemented in Relion 2.0.249. be, As in a, but for the OC1-POL3 (b), OC2-POL3 (c), oPOL3 (d) and cPOL3 (e) reconstructions.

Extended Data Figure 4 Architecture of TFIIIB and Pol III subunits involved in PIC assembly.

a, Domain architecture of TFIIIB and Pol III subunits. Protein regions are depicted according to their presence (solid colour boxes) or absence (empty boxes) in the OC-PIC structure. Regions built de novo (for which previous structural information was not available) are highlighted with a black line (full atomic model) or with a dashed black line (backbone model). The same colour scheme is used for ribbon models and cryo-EM maps in bg. b, S. cerevisiae Brf1 domain architecture. Regions absent in the density are indicated as a dashed line. C and N termini are indicated. c, Bdp1 domain architecture. Regions absent in the density are indicated as a dashed line. C and N termini are indicated. d, C37 termination–initiation loop architecture. e, C34 domain architecture. f, Clamp helices architecture. g, C31 stalk bridge architecture.

Extended Data Figure 5 Structural conservation.

a, Structure alignment of S. cerevisiae Brf1 and H. sapiens BRF2 (PDB code: 5N9G). S. cerevisiae TBP (pink) and Brf1 B-core cyclin repeats (green) are represented as molecular surfaces. Brf1 helical pin and Brf2 molecular pin are shown as green and wheat cylinders, respectively. b, Sequence alignment of S. cerevisiae Brf1 helical pin and H. sapiens BRF2 molecular pin. Residues are coloured according to their percentage identity, with dark and light blue indicating high and low sequence identity, respectively. Structurally conserved residues between the Brf1 helical pin and the Brf2 molecular pin are outlined in red. c, Multiple-sequence alignment of the clamp helices of RPA190 (Pol I), RPB1 (Pol II) and RPC160 (Pol III). Residues are coloured according to their percentage identity, with dark and light blue indicating high and low sequence identity, respectively. Residues participating in the template strand pocket (W294, L298 and Y318) are outlined in red. d, Multiple-sequence alignment of the bridge helices of RPA190 (Pol I), RPB1 (Pol II) and RPC160 (Pol III), coloured as in c. Conserved residue Y884 is outlined in red.

Extended Data Figure 6 General comparison of Pol I, Pol II and Pol III PICs.

a, Front views of S. cerevisiae Pol I PIC (PDB code: 5OA1), Pol II OC (PDB code: 5FYW) and Pol III PIC (this work). Pol III subunits are coloured as in Fig. 1. Colour scheme of Pol I and Pol II is based on architectural similarities to the Pol III system. b, Upstream DNA path differences. The DNA pathway in the Pol III PIC (light and dark blue) is different from that in yeast (wheat, PDB code: 5FYW) or human (pink, PDB code: 5IYB) Pol II PICs, probably owing to the interaction with the Bdp1 clip domain (orange). c, Comparison of Pol I, Pol II and Pol III protrusion tip in the PICs. Pol I (PDB code: 5OA1) and Pol III (this work) contact the promoter DNA through residues of the protrusion tip. Pol I participates in an extensive network of interactions that involve the binding of a α-helix to the major groove of the DNA, whereas Pol III binds to the non-template strand of the DNA (light blue) through the conserved residue K409. Pol II protrusion (PDB code: 5FYW) does not participate in direct contacts with the DNA.

Extended Data Figure 7 Mechanism of Pol III transcription initiation.

a, In unbound Pol III the stalk region and clamp are mobile and can adopt an open or closed conformation. b, Upon TFIIIB recruitment, C34 WHD1 and WHD2 are positioned over the cleft through the interaction with the Bdp1 tether and the C37 termination–initiation loop. c, The C34 WHD2 and the Brf1 N-terminal cyclin fold promote DNA melting, which occurs through stabilization of the template strand in a template strand pocket of the clamp helices, and the non-template strand between the C82 cleft loop and the C128 tip lobe domain. Contraction of the clamp helices induces a conformational change in the C82–C34–C31 subcomplex. The stalk and clamp are now locked by the C31 stalk bridge. d, The template strand is loaded in the active site and the transcription bubble is fully expanded, as binding of the Brf1 Zn-ribbon domain clears the DNA loading pathway. The template strand is correctly engaged in the active site cleft, in a configuration primed for elongation. The transcription bubble around the active site is very stable and might even be maintained in circumstances in which the main contacts with TFIIIB are disrupted. The clamp is locked in a closed conformation that prevents the re-annealing of the transcription bubble. This might be particularly important during promoter escape, as short Pol III DNA–RNA hybrids are less tightly bound than in Pol II28. The stalk and clamp are locked in a closed state that prevents bubble reannealing. e, RNA synthesis starts, the clamp helices are released and the clamp and stalk are now unlocked. The clamp remains closed during elongation but can re-open during the following steps of the transcription cycle. The rudder is repositioned and occludes access to the template strand pocket, presumably to ensure Pol III processivity.

Extended Data Figure 8 High density peaks in unbound Pol III cryo-EM reconstructions.

The globular density peaks (blue) observed in the cryo-EM maps of the unbound Pol III reconstructions are represented at three different threshold levels. The strong features of these regions are observed even at high threshold levels, suggesting the presence of metal clusters in hydrophilic–hydrophobic pockets of Pol III. Pol III core subunits are depicted in grey and the active site magnesium ion is represented as a magenta sphere.

Extended Data Table 1 Cryo-EM data collection, refinement and model statistics

Supplementary information

Life Sciences Reporting Summary (PDF 71 kb)

Conformational changes upon PIC and open complex formation

Morphing between cPol3 and OC-PIC cryo-EM structures shows the structural rearrangements occurring during PIC formation, which result in the lock of the Pol III clamp and the stabilisation of the downstream edge of the DNA bubble. The morphing and the video were generated using Chimera55. OC-PIC core subunits are depicted as grey molecular surfaces. The C82/C34/C31 subcomplex, stalk, TFIIIB subunits and C160 clamp helices are shown as ribbon and coloured as in Figure 1b. (MP4 14857 kb)

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Abascal-Palacios, G., Ramsay, E., Beuron, F. et al. Structural basis of RNA polymerase III transcription initiation. Nature 553, 301–306 (2018). https://doi.org/10.1038/nature25441

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