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- EMDB-37848: RNA polymerase II elongation complex bound with Elf1, Spt4/5 and ... -

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Basic information

Entry
Database: EMDB / ID: EMD-37848
TitleRNA polymerase II elongation complex bound with Elf1, Spt4/5 and foreign DNA, stalled at SHL(0) of the nucleosome
Map data
Sample
  • Complex: RNA polymerase II - nucleosome complex
    • Complex: RNA polymerase II
    • Complex: HistonesHistone
    • Complex: DNA, RNA
KeywordsTranscription / RNAPII / Nucleosome / Cryo-EM
Biological speciesKomagataella phaffii (fungus) / Homo sapiens (human) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 6.7 Å
AuthorsAkatsu M / Fujita R / Ogasawara M / Ehara H / Kujirai T / Takizawa Y / Sekine S / Kurumizaka H
Funding support Japan, 10 items
OrganizationGrant numberCountry
Japan Society for the Promotion of Science (JSPS)JP20H03201 Japan
Japan Society for the Promotion of Science (JSPS)JP19K06522 Japan
Japan Society for the Promotion of Science (JSPS)JP20K15711 Japan
Japan Society for the Promotion of Science (JSPS)JP18H05534 Japan
Japan Society for the Promotion of Science (JSPS)JP23H05475 Japan
Japan Society for the Promotion of Science (JSPS)JP20H05690 Japan
Japan Science and TechnologyJPMJCR16G1 Japan
Japan Science and TechnologyJPMJSP2108 Japan
Japan Agency for Medical Research and Development (AMED)JP22ama121009 Japan
Japan Science and TechnologyJPMJER1901 Japan
CitationJournal: J Biol Chem / Year: 2023
Title: Cryo-EM structures of RNA polymerase II-nucleosome complexes rewrapping transcribed DNA.
Authors: Munetaka Akatsu / Haruhiko Ehara / Tomoya Kujirai / Risa Fujita / Tomoko Ito / Ken Osumi / Mitsuo Ogasawara / Yoshimasa Takizawa / Shun-Ichi Sekine / Hitoshi Kurumizaka /
Abstract: RNA polymerase II (RNAPII) transcribes DNA wrapped in the nucleosome by stepwise pausing, especially at nucleosomal superhelical locations -5 and -1 [SHL(-5) and SHL(-1), respectively]. In the ...RNA polymerase II (RNAPII) transcribes DNA wrapped in the nucleosome by stepwise pausing, especially at nucleosomal superhelical locations -5 and -1 [SHL(-5) and SHL(-1), respectively]. In the present study, we performed cryo-electron microscopy analyses of RNAPII-nucleosome complexes paused at a major nucleosomal pausing site, SHL(-1). We determined two previously undetected structures, in which the transcribed DNA behind RNAPII is sharply kinked at the RNAPII exit tunnel and rewrapped around the nucleosomal histones in front of RNAPII by DNA looping. This DNA kink shifts the DNA orientation toward the nucleosome, and the transcribed DNA region interacts with basic amino acid residues of histones H2A, H2B, and H3 exposed by the RNAPII-mediated nucleosomal DNA peeling. The DNA loop structure was not observed in the presence of the transcription elongation factors Spt4/5 and Elf1. These RNAPII-nucleosome structures provide important information for understanding the functional relevance of DNA looping during transcription elongation in the nucleosome.
History
DepositionOct 19, 2023-
Header (metadata) releaseNov 29, 2023-
Map releaseNov 29, 2023-
UpdateDec 20, 2023-
Current statusDec 20, 2023Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_37848.png

Downloads & links

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Map

FileDownload / File: emd_37848.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.06 Å
Density
Contour LevelBy AUTHOR: 0.008
Minimum - Maximum-0.0039244425 - 0.035030957
Average (Standard dev.)0.00035251063 (±0.0020438847)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions360360360
Spacing360360360
CellA=B=C: 381.59998 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_37848_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_37848_half_map_2.map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : RNA polymerase II - nucleosome complex

EntireName: RNA polymerase II - nucleosome complex
Components
  • Complex: RNA polymerase II - nucleosome complex
    • Complex: RNA polymerase II
    • Complex: HistonesHistone
    • Complex: DNA, RNA

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Supramolecule #1: RNA polymerase II - nucleosome complex

SupramoleculeName: RNA polymerase II - nucleosome complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#22

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Supramolecule #2: RNA polymerase II

SupramoleculeName: RNA polymerase II / type: complex / ID: 2 / Parent: 1
Source (natural)Organism: Komagataella phaffii (fungus)

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Supramolecule #3: Histones

SupramoleculeName: Histones / type: complex / ID: 3 / Parent: 1
Source (natural)Organism: Homo sapiens (human)

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Supramolecule #4: DNA, RNA

SupramoleculeName: DNA, RNA / type: complex / ID: 4 / Parent: 1
Source (natural)Organism: synthetic construct (others)

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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

Concentration0.09144 mg/mL
BufferpH: 7.5
Component:
ConcentrationNameFormula
20.0 mMHEPES-KOH(pH7.5)
50.0 mMPotassium acetateCH3COOK
200.0 nMZinc acetate(CH3COO)2Zn
0.1 mMTCEP-HCl

Details: 20 mM HEPES-KOH(pH7.5), 50 mM Potassium acetate, 200 nM Zinc acetate, 0.1 mM TCEP-HCl
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK III
DetailsdsDNA concentration is 0.09144 mg/mL. This sample contains 0.005% Tween20.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.0 µm
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 59.84 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: EMDB MAP
EMDB ID:

9713

Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionResolution.type: BY AUTHOR / Resolution: 6.7 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 12176

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