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Structural basis of the complete poxvirus transcription initiation process

Nature Structural & Molecular Biology volume 28, pages 779–788 (2021)Cite this article

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Abstract

Poxviruses express their genes in the cytoplasm of infected cells using a virus-encoded multi-subunit polymerase (vRNAP) and unique transcription factors. We present cryo-EM structures that uncover the complete transcription initiation phase of the poxvirus vaccinia. In the pre-initiation complex, the heterodimeric early transcription factor VETFs/l adopts an arc-like shape spanning the polymerase cleft and anchoring upstream and downstream promoter elements. VETFI emerges as a TBP-like protein that inserts asymmetrically into the DNA major groove, triggers DNA melting, ensures promoter recognition and enforces transcription directionality. The helicase VETFs fosters promoter melting and the phospho-peptide domain (PPD) of vRNAP subunit Rpo30 enables transcription initiation. An unprecedented upstream promoter scrunching mechanism assisted by the helicase NPH-I probably fosters promoter escape and transition into elongation. Our structures shed light on unique mechanisms of poxviral gene expression and aid the understanding of thus far unexplained universal principles in transcription.

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Fig. 1: Structure of the vaccinia PIC.
Fig. 2: Structure of the VETF heterodimer.
Fig. 3: Comparison of the TBP-like domain from vaccinia VETFl with yTBP.
Fig. 4: Structure of the late PIC.
Fig. 5: Three structures of initially transcribing complexes.
Fig. 6: Structure of the lITC.
Fig. 7: Transition of complete vRNAP to the PIC, and a model for early promoter recognition and opening.

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Data availability

Crystallographic structure factors and atomic coordinates have been deposited in the Protein Data Bank (PDB) under accession nos. 7AMV, 7AOF, 7AOZ, 7AP8, 7AP9 and 7AOH. Cryo-EM maps have been deposited with the Electron Microscopy Data Bank (EMDB) under accession nos. EMD-11824, EMD-11843, EMD-11848, EMD-11850, EMD-11851 and EMD-11844. Source data are provided with this paper.

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Acknowledgements

We thank C. Kraft for help during cryo-EM data collection. Cryo-EM of the complete vRNAP was carried out in the cryo-EM facility of the University of Würzburg (DFG – INST 93/903-1 FUGG). This work was supported by the DFG (Fi 573/21-1) and the Hope Realized Medical Foundation.

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Author notes

  1. These authors contributed equally: Clemens Grimm, Julia Bartuli.

Authors and Affiliations

  1. Department of Biochemistry and Cancer Therapy Research Center (CTRC), Theodor Boveri-Institute, University of Würzburg, Würzburg, Germany

    Clemens Grimm, Julia Bartuli, Aladar A. Szalay & Utz Fischer

  2. Department of Biochemistry, Rudolf-Virchow-Center, University of Würzburg, Würzburg, Germany

    Bettina Boettcher

  3. Helmholtz Institute for RNA-based Infection Research (HIRI), Helmholtz Centre for Infection Research (HZI), Würzburg, Germany

    Aladar A. Szalay & Utz Fischer

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  1. Clemens Grimm
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Contributions

J.B. and C.G. designed transcription scaffolds and acquired cryo-EM data. B.B. assisted during cryo-EM data collection. J.B. performed functional vRNAP and scaffold binding assays, purified the vRNAP complexes and prepared cryo-EM samples. C.G. processed the cryo-EM data, built and refined the models, analyzed the models and prepared the figures and videos. C.G. and U.F. wrote the manuscript. U.F. designed and supervised the project. A.A.S. and U.F. acquired funding.

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Correspondence to Clemens Grimm or Utz Fischer.

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Extended data

Extended Data Fig. 1 Reconstitution and preparative purification of vRNAP-promoter complexes.

Approx. 400 µg affinity-purified complete vRNAP was incubated with a 60-fold molar excess of the synthetic DNA scaffold in the presence of 1 mM NTPs and separated by gradient centrifugation. Fractions 13-16 were pooled and used for cryo-EM studies. (b) Representative micrograph and (c) representative 2D classes of the dataset. If applicable, the images are annotated with the names of the predominant particle type. (d) Classification and refinement scheme. (e) Local resolution mapped to the consensus reconstruction density iso-surface (only a mild B-factor sharpening of -10 Å2 was applied). (f) Masked VETF and DNA region after multibody refinement. (g) FSC curves for consensus and multibody refinements. (h) Angular distribution plots referring to the consensus reconstruction in E. (i) Selected views of the final, B-factor sharpened (−60 Å2) cryo-EM density isosurface overlaid with the model.

Extended Data Fig. 2 DNA contacts in the PIC.

Transparent blue iso-surface of the cryo-EM density for the bound DNA, filtered by a Gaussian blur to 1.5σ standard deviation. The model is shown in cartoon style and the initially melted region (IMR) is indicated. Top view of the PIC with VETF removed (top view) and vRNAP core shown as solvent accessible surface. The clamp head and lobe are marked on the molecular surface by a rose dotted line, respectively. (b) Front view of the PIC with core removed (front view) and VETF shown in cartoon representation. (c) PIC with vRNAP removed shown in cartoon view turned by roughly 90° relative to B. and slightly optimized for clarity (d) Upstream promoter contacts to the core vRNAP. Detailed view of the upstream promoter contacts to the core vRNAP in cartoon representation. The lobe region contacting the DNA is indicated with a rose dotted line. (e) Cartoon model of VETFs and downstream DNA with superposed ideal B DNA in transparent grey is shown. The respective helix axes are indicated and Phe 271 is depicted in stick representation. (f) A depiction of the promoter-bound yeast XPB homologue SSL2 from the yeast PIC bound to TFIIH and core mediator (Schilbach et al., 2017) (PDB:5oqm) analogous to a. The axis of the bent, bound DNA (blue) is indicated. Both arms of the DNA helix axis bend angles in (e) and (f) lie approximately in the paper plane. (g) Vaccinia PIC model in cartoon representation as shown in Fig. 1a, front view. (h) Pol II core PIC model (PDB 5IY6) in cartoon representation and oriented by superposition of the Pol II core polymerase with the core vRNAP of the vaccinia PIC in (g). Elements identified as functionally, architecturally or structurally corresponding are colored according to the scheme used for the vaccinia PIC throughout this manuscript.

Extended Data Fig. 3 Cryo EM reconstruction of lPIC and ITC.

(a) classification and refinement scheme. Areas used for local classification is encircled in magenta. (b) Local resolution mapped to the reconstruction density isosurface. (c) FSC plots for consensus refinement and the separaty bodies of the multibody (MB) refinement. (d) Angular orientation plots referring to the reconstructions in (b).

Extended Data Fig. 4 Cryo EM reconstruction of the lITC.

(a) classification and refinement scheme. (b) Local resolution mapped to the reconstruction density isosurface. (c) FSC plots for consensus refinement and the two-bogy MB refinement, respectively. (d) Orientation plot referring to the consensus reconstruction in (b).

Extended Data Fig. 5 Details of the DNA in the ITC and lITC.

(a) Cryo EM density for DNA in the ITC. (b) Cryo EM density for DNA in the lITC. The blunt ends of the synthetic DNA scaffold are recognizable in the density. Note that the position of the downstream end of the scaffold has advanced roughly 5 bp when comparing the ITC (a) to the lITC (b) structure. The visible upstream blunt end indicates extensive promoter scrunching. (c) Transcription bubble in the lITC. A zoomed view into the active site region is depicted. Disordered regions of the template and non-template strand are shown as dotted lines. The start and end positions of the melted promoter and the base at the active site are numbered relative to the TSS. (d) illustration of major rearrngments during ITC formation. The lITC model is shown in carton representation. The B-cyclin as in the lPIC structure is shown as a molecular surface. In this orientation it would obstruct the upstream DNA path. The domain rearrangement is indicated ba a magenta arrow. (e) Analogous to (d), the B ribbon as in the lPIC structure is shown as a molecular surface, its rearrangement indicated as a magenta arrow. The stabilization of transiently flexible regions of Rap94 in the lITC by formation of a β strand to the core vRNAP is highlighted with a magenta box.

Extended Data Fig. 6 Comparison of Vaccinia NPH-I and VETFs with structurally related helicases.

Color code according to common structural elements. The helicase ATPase modules were extracted from structures with the following PDB ID codes: Complete vRNAP: 6RFL, INO80: 6HTS, Rad26: 5VVR, XPB: 6NMI.

Supplementary information

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Supplementary Video 1

Reconfiguration of complete vRNAP to the PIC and details of promoter binding and melting.

Supplementary Video 2

Remodeling of the vaccinia PIC, the template strand capture mechanism and upstream promoter scrunching.

Source data

Source Data Fig. 1

Scan of film for the band shift experiment.

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Grimm, C., Bartuli, J., Boettcher, B. et al. Structural basis of the complete poxvirus transcription initiation process. Nat Struct Mol Biol 28, 779–788 (2021). https://doi.org/10.1038/s41594-021-00655-w

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