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- EMDB-15358: Structure of dimeric yeast RNA polymerase II bound to a transcrip... -

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Basic information

Entry
Database: EMDB / ID: EMD-15358
TitleStructure of dimeric yeast RNA polymerase II bound to a transcription bubble (consensus map)
Map dataRNA Polymerase II dimer loaded with a DNA-RNA scaffold
Sample
  • Complex: S. cerevisiae RNA polymerase II
Keywordstranscription regulation / RNA 3' end processing / transcription termination / GENE REGULATION
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.6 Å
AuthorsCarminati M / Manav MC / Bellini D / Passmore LA
Funding supportEuropean Union, 2 items
OrganizationGrant numberCountry
European Commission725685European Union
European Molecular Biology Organization (EMBO)556-2018European Union
CitationJournal: Mol Cell / Year: 2023
Title: A direct interaction between CPF and RNA Pol II links RNA 3' end processing to transcription.
Authors: Manuel Carminati / Juan B Rodríguez-Molina / M Cemre Manav / Dom Bellini / Lori A Passmore /
Abstract: Transcription termination by RNA polymerase II (RNA Pol II) is linked to RNA 3' end processing by the cleavage and polyadenylation factor (CPF or CPSF). CPF contains endonuclease, poly(A) polymerase, ...Transcription termination by RNA polymerase II (RNA Pol II) is linked to RNA 3' end processing by the cleavage and polyadenylation factor (CPF or CPSF). CPF contains endonuclease, poly(A) polymerase, and protein phosphatase activities, which cleave and polyadenylate pre-mRNAs and dephosphorylate RNA Pol II to control transcription. Exactly how the RNA 3' end processing machinery is coupled to transcription remains unclear. Here, we combine in vitro reconstitution, structural studies, and genome-wide analyses to show that yeast CPF physically and functionally interacts with RNA Pol II. Surprisingly, CPF-mediated dephosphorylation promotes the formation of an RNA Pol II stalk-to-stalk homodimer in vitro. This dimer is compatible with transcription but not with the binding of transcription elongation factors. Disruption of the dimerization interface in cells causes transcription defects, including altered RNA Pol II abundance on protein-coding genes, tRNA genes, and intergenic regions. We hypothesize that RNA Pol II dimerization may provide a mechanistic basis for the allosteric model of transcription termination.
History
DepositionJul 7, 2022-
Header (metadata) releaseNov 8, 2023-
Map releaseNov 8, 2023-
UpdateJan 10, 2024-
Current statusJan 10, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_15358.png

Downloads & links

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Map

FileDownload / File: emd_15358.map.gz / Format: CCP4 / Size: 476.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationRNA Polymerase II dimer loaded with a DNA-RNA scaffold
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 500 pix.
= 415. Å		0.83 Å/pix.
x 500 pix.
= 415. Å		0.83 Å/pix.
x 500 pix.
= 415. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.83 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.008
Minimum - Maximum-0.007801991 - 0.02486588
Average (Standard dev.)0.0002527381 (±0.0013749115)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions500500500
Spacing500500500
CellA=B=C: 415.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_15358_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_15358_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : S. cerevisiae RNA polymerase II

EntireName: S. cerevisiae RNA polymerase II
Components
  • Complex: S. cerevisiae RNA polymerase II

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Supramolecule #1: S. cerevisiae RNA polymerase II

SupramoleculeName: S. cerevisiae RNA polymerase II / type: complex / ID: 1 / Parent: 0
Details: RNA polymerase 'stalk-to-stalk' homodimer loaded with a DNA-RNA scaffold mimicking a transcription bubble
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 552 KDa

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration1.1 mg/mL
BufferpH: 8
Component:
ConcentrationName
10.0 mMK-HEPES
100.0 mMKCl
0.5 mMTCEP

Details: 0.005 % v/v Tween-20 was added to the sample just before vitrification to prevent preferred orientation problems.
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Time: 45 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV / Details: blot for 4 seconds (force -12) before plunging.
DetailsThe vitrified sample contained Pol II (with transcription bubble) bound to Ref2:Glc7:Swd2. We observed a dimeric Pol II population (~10 % of particles) which is reported in the present deposition.

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Electron microscopy #1

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.7 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 105000
Specialist opticsEnergy filter - Slit width: 20 eV
Sample stageCooling holder cryogen: NITROGEN
Microscopy ID1
DetailsThe movies were collected without tilt or with a tilt angle over a 33-45 degrees range.
Image recordingImage recording ID: 1 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number grids imaged: 6 / Number real images: 25411 / Average electron dose: 40.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Electron microscopy #1~

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.7 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 75000
Sample stageCooling holder cryogen: NITROGEN
Microscopy ID1
DetailsThe movies were collected without tilt or with a tilt angle over a 30-40 degrees range.
Image recordingImage recording ID: 2 / Film or detector model: FEI FALCON III (4k x 4k) / Detector mode: COUNTING / Number grids imaged: 2 / Number real images: 2035 / Average electron dose: 40.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 5000000
Details: 800000 particles from the Falcon II datasets were used for the final reconstruction (please refer to the methods in the paper)
Startup modelType of model: EMDB MAP
EMDB ID:

8736

Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final 3D classificationNumber classes: 6 / Software - Name: RELION (ver. 3.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 4.6 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1)
Details: Consensus refined map of homodimeric RNA Pol II before focused refinement on monomer 1 (EMD-15359) or monomer 2 (EMD-15360).
Number images used: 151000
DetailsConsensus (refined) map of the intact Pol II dimer before focused refinement on monomer 1 (EMD-15359) and monomer 2 (EMD-15360).
Image recording ID1
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

5c4x


Chain - Source name: PDB / Chain - Initial model type: experimental model
DetailsTwo copies of the monomeric Pol II structure (PDB: 5C4X) were rigid fit into the dimeric Pol II EM density
RefinementProtocol: RIGID BODY FIT

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