Structural insights into the nucleic acid remodeling mechanisms of the yeast THO-Sub2 complex
- Department of Structural Cell Biology, Max Planck Institute of Biochemistry, Germany
Figures
Figure 1
Biochemical reconstitution and native isolation of Saccharomyces cerevisiae THO-Sub2.
(A) Domain organization of Tho2 (yellow), Hpr1 (green), Mft1 (light blue), Thp2 (dark blue), Tex1 (cyan), and Sub2 (with the RecA1 domain in pink and the RecA2 domain in purple). Gray parts are not …
Figure 2 with 4 supplements
Cryo-electron microscopy (cryo-EM) reconstruction of Saccharomyces cerevisiae THO-Sub2 homodimer.
(A) Segmented cryo-EM reconstruction of the THO-Sub2 dimer. Three different views are shown; proteins and domains are colored as in Figure 1A. Features discussed in the text are indicated, including …
Figure 2—figure supplement 1
Cryo-electron microscopy analysis.
(A) Representative micrograph acquired on a 300 keV Titan Krios equipped with a post-GIF K3 detector (Gatan, 5760 × 4092 pixel) showing particle distribution and reference-free 2D class-averages. (B)…
Figure 2—figure supplement 2
High-resolution THO-Sub2 density.
(A) Segmented cryo-electron microscopy reconstruction of the rigid THO-Sub2 protomer used for de novo atomic modeling. Front and back views are shown; proteins and domains are colored as in Figure 2.…
Figure 2—figure supplement 3
Model quality.
Left: Ramachandran plot of the main-chain φ and ψ torsional angles of the rigid protomer THO-Sub2 atomic model. Areas of favored φ and ψ combinations are defined in dark blue (see also Supplementary …
Figure 2—figure supplement 4
Comparison of our cryo-electron microscopy structure with previous structural studies.
(A) Comparison with a previous negative-stain analyses of the THO complex (Peña et al., 2012; Ren et al., 2017). Arrows highlight areas with distinctive features in the 2D class-averages and our …
Figure 3 with 5 supplements
THO complex is built from intertwined conserved interactions.
(A) Front view of the THO-Sub2 rigid protomer shown as a cartoon backbone representation. (B) Same view as A with the five modules of the Tho2-Hpr1 platform in different colors. The rectangles …
Figure 3—figure supplement 1
Structural features of the THO complex.
Figure 3—figure supplement 2
Tho2 structure-based sequence alignment.
The sequence alignment includes Tho2 orthologues from Saccharomyces cerevisiae (P53552), Schizosaccharomyces pombe (Q09779), Homo sapiens (Q8NI27), Danio rerio (F1R5B5), and Drosophila melanogaster …
Figure 3—figure supplement 3
Hpr1 structure-based sequence alignment.
The sequence alignment includes the structured elements of Hpr1 orthologues from Saccharomyces cerevisiae (P17629), Schizosaccharomyces pombe (Q9URT2), Homo sapiens (Q96FV9), Danio rerio (Q7SYB2), …
Figure 3—figure supplement 4
Tex1 structure-based sequence alignment.
The sequence alignment includes Tex1 orthologues from Saccharomyces cerevisiae (P53851), Schizosaccharomyces pombe (Q9USL1), Homo sapiens (Q96J01), Danio rerio (Q6AXK9), and Drosophila melanogaster …
Figure 3—figure supplement 5
Sub2 structure-based sequence alignment.
The sequence alignment includes Sub2 orthologues from Saccharomyces cerevisiae (Q07478), Schizosaccharomyces pombe (O13792), Homo sapiens (Q13838), Danio rerio (Q7SXU7), and Drosophila melanogaster …
Figure 4 with 1 supplement
THO homodimerization properties.
The central panel shows the back and front views of the THO-Sub2 homodimer, with the whole complex in gray except the dimerization elements highlighted in color: the two Mft1-Thp2 protomers (back …
Figure 4—figure supplement 1
Superposition of the two THO protomers.
The flexible protomer (colored in gray) is aligned on the rigid protomer (colored as in Figure 2). The coiled-coils of Mft1-Thp2 start to bend at the point where the Tho2 β-hairpin is close to Mft1 …
Figure 5 with 1 supplement
Sub2-activated conformation at the proximal side of the THO homodimer.
(A) Sub2-Tho2 interaction at the proximal side. The zoom views show a subset of conserved interacting residues. See also Figure 3—figure supplements 2 and 5. (B) Structure of DDX6-CNOT1-4ET (Ozgur …
Figure 5—figure supplement 1
Conformational states of Sub2.
Comparison of the activated semi-closed Sub2 structure as found at the proximal side of the THO homodimer cryo-electron microscopy structure with the conformation of active Sub2, bound to RNA, …
Figure 6 with 1 supplement
Hypothetical model of transcription-export (TREX) molecular mechanisms.
Schematic depicts the TREX complex: on the left at resting state, with Yra1 bound to the Sub2 RecA1 domains via its N-box and C-box motifs (Ren et al., 2017); on the right in a substrate-binding …
Figure 6—figure supplement 1
Hypothetical model of THO-Sub2-Yra1 (transcription-export) in binding RNA:DNA hybrids.
(A) The scheme on the top left shows the domain organization of Yra1. The N-terminal and C-terminal regions of Yra1 bind to Sub2 with similar affinities as measured in the fluorescence anisotropy …
Videos
Video 1
Dynamic character of the complex extracted from cryo-electron microscopy data.
Variance analysis of the THO-Sub2 complex structure, showing the swiveling motion of the two protomers with respect to each other.
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (Escherichia coli) | BL21 Star (DE3) pRARE | EMBL Heidelberg Core Facility | Electrocompetent cells | |
| Cell line (Spodoptera frugiperda) | IPLB-Sf21-AE | Gibco | ||
| Cell line (Trichoplusia ni) | BTI-Tn-5B1-4 | Gibco | ||
| Strain, strain background (Saccharomyces cerevisiae) | BY4741 (MATa) yeast | Euroscarf | Y00000 | |
| Strain, strain background (Saccharomyces cerevisiae) | BY4743 (MATa/α) yeast | Euroscarf | Y20000 | |
| Antibody | Anti-protein-A IgG (mouse, monoclonal) | Sigma Aldrich | P2921 | (1:333 dilution) |
| Recombinant DNA reagent | Tho2 | This paper (Materials and methods) | Uniprot P53552 | pFastBac Hta-Tho2 Conti Lab |
| Recombinant DNA reagent | Hpr1 | This paper (Materials and methods) | Uniprot P17629 | pFastBac Hta-Hpr1 Conti Lab |
| Recombinant DNA reagent | Mft1 | This paper (Materials and methods) | Uniprot P33441 | pFastBac Hta-Mft1 Conti Lab |
| Recombinant DNA reagent | Thp2 | This paper (Materials and methods) | Uniprot O13539 | pFastBac Hta-Thp2 Conti Lab |
| Recombinant DNA reagent | Tex1 | This paper (Materials and methods) | Uniprot P53851 | pFastBac Hta-Tex1 Conti Lab |
| Recombinant DNA reagent | Sub2 | This paper (Materials and methods) | Uniprot Q07478 | 3C-GST-fusion Conti Lab |
| Recombinant DNA reagent | GFP-Sub2 | This paper (Materials and methods) | Conti Lab | |
| Commercial assay or kit | Bac-to-Bac Baculovirus Expression System | ThermoFisher Scientific | ||
| Software, algorithm | SerialEM | https://bio3d.colorado.edu/SerialEM/ | SerialEM_3-8- 0beta8_64 & SerialEM_3-8- 0beta11_64 | |
| Software, algorithm | Focus | https://focus.c-cina.unibas.ch/ wiki/doku.php | v 1.1.0 | |
| Software, algorithm | cryosparc | doi: 10.1038/nmeth.4169 | Cryosparc2 | |
| Software, algorithm | CTFfind4 | doi: 10.1016/j.jsb.2015.08.008 | ||
| Software, algorithm | TOPAZ | doi: 10.1038/s41592-019-0575-8 | ||
| Software, algorithm | UCSF Chimera | UCSF, https://www.cgl.ucsf.edu/chimera/ | ||
| Software, algorithm | UCSF ChimeraX | UCSF, https://www.rbvi.ucsf.edu/chimerax/ | ||
| Software, algorithm | COOT | http://www2.mrc-lmb.cam.ac.uk/ personal/pemsley/coot/ | 0.9 | |
| Software, algorithm | Phenix | https://www.phenix-online.org/ | PHENIX 1.18 | |
| Software, algorithm | Molprobity | Duke Biochemistry, http://molprobity.biochem.duke.edu/ | ||
| Software, algorithm | PyMol 2 | PyMOL Molecular Graphics System, Schrodinger LLC | PyMOL 2.1 |
Additional files
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Supplementary file 1
Cryo-electron microscopy data collection, refinement, and validation statistics.
- https://cdn.elifesciences.org/articles/61467/elife-61467-supp1-v2.docx
-
Transparent reporting form
- https://cdn.elifesciences.org/articles/61467/elife-61467-transrepform-v2.docx
