Molecular mechanism of promoter opening by RNA polymerase III

Abstract

RNA polymerase III (Pol III) and transcription factor IIIB (TFIIIB) assemble together on different promoter types to initiate the transcription of small, structured RNAs. Here we present structures of Pol III preinitiation complexes, comprising the 17-subunit Pol III and the heterotrimeric transcription factor TFIIIB, bound to a natural promoter in different functional states. Electron cryo-microscopy reconstructions, varying from 3.7 Å to 5.5 Å resolution, include two early intermediates in which the DNA duplex is closed, an open DNA complex, and an initially transcribing complex with RNA in the active site. Our structures reveal an extremely tight, multivalent interaction between TFIIIB and promoter DNA, and explain how TFIIIB recruits Pol III. Together, TFIIIB and Pol III subunit C37 activate the intrinsic transcription factor-like activity of the Pol III-specific heterotrimer to initiate the melting of double-stranded DNA, in a mechanism similar to that of the Pol II system.

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Figure 1: Cryo-EM structure of the Pol III PIC.
Figure 2: Interactions of TFIIIB with C34 and C37 in the PIC.
Figure 3: Protein–DNA contacts in the Pol III PIC.
Figure 4: Comparison between Pol III CC and OC structures.
Figure 5: Mechanism of promoter opening and DNA melting in Pol III.

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Acknowledgements

C.W.M. and R.W. acknowledge support from the European Research Council Advanced Grant (ERC-2013-AdG340964-POL1PIC). M.K.V. acknowledges support from the EMBL International PhD program. H.K. acknowledges support from the European Union’s Horizon 2020 program under the Marie Sklodowska-Curie grant agreement no. 703432. We thank F. Baudin for help with the transcription assay, A. J. Jakobi for extensive help with setting up LocScaling of cryo-EM maps and model refinement, T. Hoffmann and J. Pecar for setting up the high performance computing for RELION 2.0, and N. A. Hoffmann, J. Kosinski, L. Tafur and Y. Sadian for discussions. We thank A. Vannini for giving us access to the coordinates of PDB entry 5N9G before publication.

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C.W.M. initiated and supervised the project. M.K.V. designed and carried out experiments, data processing and model building. H.K. helped in the initial stages of electron microscopy preparation. W.J.H.H. collected cryo-EM datasets. R.W. was responsible for yeast fermentation and helped with cloning. M.K.V. and C.W.M. prepared the manuscript with input from the other authors.

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Correspondence to Christoph W. Müller.

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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 analysis of the Pol III PIC.

a, Cryo-EM densities sharpened with relion_postprocess of the Pol III ITC, OC, CC1 and CC2 maps reported here superimposed with the corresponding models. b, Typical micrograph of the Pol III PIC. c, Fourier-shell correlation curves for the different cryo-EM maps, as reported by the relion_postprocess program.

Extended Data Figure 2 Classification strategies for cryo-EM datasets.

Left, ITC dataset. The ITC dataset was cleaned (step 1) by classification in 3D (binned 4 times) and PIC particles were selected using a mask on TFIIIB (step 2). Finally, classification using a mask on downstream DNA yielded the ITC and the OCΔdownstream−1 maps (step 3). Right, CC datasets. Two CC datasets were individually cleaned (step 1) by classification in 3D (binned 4 times) and PIC particles were selected using a mask on TFIIIB (step 2). PIC particles from both datasets were combined and classified without masking to remove a small subset of dimers (step 3). Classification using a mask on C34 WH1 and WH2 yielded the OC and CC populations (step 4) which were subsequently classified into CC1, CC2, OC and OCΔdownstream−2 (step 5). The majority of particles from the CC datasets are in the OC state (67% of PIC particles, versus 32% in the CC states, Extended Data Fig. 2), showing that our TFIIIB–Pol III complex is active in promoter opening. Focused classification on downstream DNA in the ITC and CC datasets gave rise to reconstructions which show OC-like upstream DNA but lack downstream DNA (OCΔdownstream). This may suggest the existence of an initiation intermediate in which downstream DNA is mobile and becomes ordered only in a later stage of the transcription cycle, as has also been described for bacterial RNA polymerase48. An alternative explanation for the lack of density in the OCΔdownstream map derived from the CC scaffold lies in the pseudo-symmetric nature of the U6 TATA box28. Because we used a wild-type promoter sequence, a subset of particles may have bound the DNA scaffold with reverse polarity, making the ‘downstream’ DNA too short to be stabilized in the cleft. However, we favour the former explanation, as we also observe OCΔdownstream in the ITC dataset, in which the polarity is defined by incubating Pol III with the preformed transcription bubble.

Extended Data Figure 3 Local resolution, Euler angular distribution and side chain densities.

a, Cryo-EM maps coloured by local resolution from 3 Å (blue) to 10 Å (red). All maps are shown on the same contour level. The central slices show that the local resolution degrades more strongly in the CC2 map compared to the ITC map, although they have comparable overall resolutions. Euler angular distribution plotting shows a good angular coverage without a dominating preferred orientation in all reconstructions. b, Examples of helical and β-strand densities in Pol III subunit AC40 in the 3.7 Å OC map.

Extended Data Figure 4 Cryo-EM density for newly modelled regions.

Cryo-EM densities in the ITC map of Bdp1 (top), C34 WH1 and WH2 (bottom left), and Brf1 (bottom right) after amplitude scaling contoured at a level that allows visualization of most residues. Top right, side-chain models and cryo-EM densities for de novo built regions.

Extended Data Figure 5 Bdp1 partially masks binding sites for TFIIIC in Brf1.

Brf1 regions that have been mapped to interact with TFIIIC are shown in yellow29. Sites 2 and 3 are partially buried by Bdp1 in the PIC. Right, close-up views of site 3 (top) and site 2 (bottom). The parts of Bdp1 shown as transparent are those that could be built but were not included in the final model owing to lack of sequence register and weak density. Bdp1 appears to compete for the same binding sites on Brf1 as TFIIIC, suggesting that the correct assembly of TFIIIB might trigger the conformational rearrangements in the assembly factor TFIIIC that render the complex initiation-competent.

Extended Data Figure 6 Comparison between Pol III in the OC and elongating states.

Superposition of elongating Pol III (ePol, PDB: 5FJ8) and Pol III in the OC. In the OC, the heterotrimer moves towards upstream DNA and the C34 WH1 and WH2 domains become ordered. The stalk moves towards the heterotrimer, and the clamp moves to slightly close the cleft. The C37 initiation/termination loop becomes partially ordered to interact with the Bdp1 tether and C34 WH1 and WH2.

Extended Data Figure 7 Active site and nucleic acid density in the ITC.

a, Cryo-EM density in the ITC map after amplitude scaling of active-site elements and nucleic acids. Elements contoured at a lower threshold are shown as mesh. Active-site elements are labelled. The rudder and trigger loops are disordered. The poor density of RNA suggests flexibility or partial occupancy due to dissociation during sample preparation or cleavage by C11. b, RNA extension assay of the 32P-labelled 6-nt RNA in the absence of CTP, showing that our preparation is active in transcription using our ITC scaffold. Addition of TFIIIB leads to a stronger accumulation of 11-nt RNA. Modelling of an elongated RNA oligonucleotide based on RNA polymerase I elongation complex 1 (PDB: 5M5X) shows that RNA clashes at position +7 with the beginning of the flexible B-linker, and at position +13 with the B-ribbon. Accumulation of 11-nt RNA in the presence of TFIIIB suggests that the RNA in the Pol III PIC takes a different path compared to that in the Pol I elongation complex, and clashes with the B-ribbon at position +12, requiring the release of TFIIIB to enter elongation. The experiment was repeated three times independently with the same results.

Extended Data Figure 8 Comparison of Pol III, Pol II and Pol I PICs.

a, Ribbon diagrams of yeast Pol III, Pol II (PDB: 5FYW) and Pol I (PDB: 5W65) PICs. TFIIF and TFIIE occupy similar positions to the C53–C37 heterodimer and the C82–C34–C31 heterotrimer, respectively. The convex surface of TBP is closely contacted by Brf1 homology domain II in TFIIIB, but is accessible in the Pol II PIC, whereas TBP is entirely absent from available structures of Pol I PIC. This might explain the strict requirement for TBP in the Pol III system, in contrast to Pol II and Pol I. b, Close-up view of the downstream promoter assembly, showing C82 WH3–WH4, C34 WH1–WH3 (left), the WH domains of TFIIF and TFIIE (middle) and the A49 tandem WH9 and TPR of Rrn11 (right). Whereas C82–C34 and TFIIF–TFIIE form structurally similar downstream promoter assemblies using WH domains and the C82 ‘cleft loop’ and TFIIE ‘E-wing’ to contact the upstream bubble edge, the Pol I core factor forms a structurally different assembly in which the A49 tandem WH does not contact the upstream bubble edge in the same way. c, Comparison of the cyclin folds in Brf1, TFIIB and Rrn7. The cyclin folds in Brf1 and TFIIB occupy similar positions and contact the polymerase wall, whereas the cyclin folds in Rrn7 do not.

Extended Data Figure 9 Comparison between Bdp1 in the Pol III PIC and TFIIA and TFIIF in the Pol II PIC.

The Bdp1 SANT domain is located at a similar position to TFIIA. Parts of Bdp1 resemble TFIIF subunit Tfg1, although no sequence similarity is detectable. Both interact with the Pol protrusion by adding a β-strand to it (although at different ends of the protrusion β-sheet) and folding into a short helix along the face of the protrusion. The path of the C37 initiation/termination loop is also similar to that of Tfg1. The second subunit of TFIIF, Tfg2, also adds a β-strand to the Pol II protrusion. Brf1 and TBP were omitted for clarity.

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

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Mechanism of promoter opening by RNA Pol III

The video shows the proposed model of promoter opening including the transition between the structures reported in this work and the modelled “closed complex/open clamp” intermediate. (MP4 25364 kb)

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Vorländer, M., Khatter, H., Wetzel, R. et al. Molecular mechanism of promoter opening by RNA polymerase III. Nature 553, 295–300 (2018). https://doi.org/10.1038/nature25440

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