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- EMDB-3382: Transcription initiation complex structures elucidate DNA opening... -

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Basic information

Entry
Database: EMDB / ID: EMD-3382
TitleTranscription initiation complex structures elucidate DNA opening (OC5)
Map data
Sampleyeast Pol II transcription initiation complex (open DNA)
  • DNA-directed RNA polymerase IIPolymerase
  • (Transcription Factor ...) x 4
  • TATA-box-binding protein
  • nucleic-acidNucleic acid
KeywordsGene expression / Transcription initiation
Biological speciesSaccharomyces cerevisiae (baker's yeast) / unidentified (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 6.1 Å
AuthorsPlaschka C / Hantsche M / Dienemann C / Burzinski C / Plitzko J / Cramer P
CitationJournal: Nature / Year: 2016
Title: Transcription initiation complex structures elucidate DNA opening.
Authors: C Plaschka / M Hantsche / C Dienemann / C Burzinski / J Plitzko / P Cramer /
Abstract: Transcription of eukaryotic protein-coding genes begins with assembly of the RNA polymerase (Pol) II initiation complex and promoter DNA opening. Here we report cryo-electron microscopy (cryo-EM) ...Transcription of eukaryotic protein-coding genes begins with assembly of the RNA polymerase (Pol) II initiation complex and promoter DNA opening. Here we report cryo-electron microscopy (cryo-EM) structures of yeast initiation complexes containing closed and open DNA at resolutions of 8.8 Å and 3.6 Å, respectively. DNA is positioned and retained over the Pol II cleft by a network of interactions between the TATA-box-binding protein TBP and transcription factors TFIIA, TFIIB, TFIIE, and TFIIF. DNA opening occurs around the tip of the Pol II clamp and the TFIIE 'extended winged helix' domain, and can occur in the absence of TFIIH. Loading of the DNA template strand into the active centre may be facilitated by movements of obstructing protein elements triggered by allosteric binding of the TFIIE 'E-ribbon' domain. The results suggest a unified model for transcription initiation with a key event, the trapping of open promoter DNA by extended protein-protein and protein-DNA contacts.
History
Current status-Processing site: PDBe / Status: Released
DepositionMar 9, 2016-
Header (metadata) releaseApr 13, 2016-
Map releaseMay 18, 2016-
UpdateJun 15, 2016-

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0125
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.0125
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_3382.map.gz / Format: CCP4 / Size: 100.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.35 Å/pix.
x 300 pix.
= 405. Å
1.35 Å/pix.
x 300 pix.
= 405. Å
1.35 Å/pix.
x 300 pix.
= 405. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.35 Å
Density
Contour LevelBy AUTHOR: 0.0125 / Movie #1: 0.0125
Minimum - Maximum-0.02028922 - 0.06281367
Average (Standard dev.)0.00012642 (±0.00241288)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 405.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.351.351.35
M x/y/z300300300
origin x/y/z0.0000.0000.000
length x/y/z405.000405.000405.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS300300300
D min/max/mean-0.0200.0630.000

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Supplemental data

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Supplemental map: pp 3dref-run23 0113 57 0B 1.map

Filepp_3dref-run23_0113_57_0B_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Sample components

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Entire yeast Pol II transcription initiation complex (open DNA)

EntireName: yeast Pol II transcription initiation complex (open DNA)
Number of components: 7 / Oligomeric State: Monomer
MassTheoretical: 890 kDa

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Component #1: protein, DNA-directed RNA polymerase II

ProteinName: DNA-directed RNA polymerase IIPolymerase / a.k.a: RNA polymerase II / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 513 kDa
SourceSpecies: Saccharomyces cerevisiae (baker's yeast) / Strain: BJ5464 Rpb3 His-Bio

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Component #2: protein, Transcription Factor IIA

ProteinName: Transcription Factor IIA / a.k.a: TFIIATranscription factor II A / Number of Copies: 1 / Recombinant expression: Yes
MassTheoretical: 45 kDa
SourceSpecies: Saccharomyces cerevisiae (baker's yeast)
Source (engineered)Expression System: Escherichia coli (E. coli) / Vector: pOPINE / Strain: BL21(DE3)RIL

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Component #3: protein, Transcription Factor IIB

ProteinName: Transcription Factor IIBTranscription factor II B / a.k.a: TFIIBTranscription factor II B / Recombinant expression: Yes / Number of Copies: 1
MassTheoretical: 38 kDa
SourceSpecies: Saccharomyces cerevisiae (baker's yeast)
Source (engineered)Expression System: Escherichia coli (E. coli) / Vector: pET21b / Strain: BL21(DE3)RIL

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Component #4: protein, TATA-box-binding protein

ProteinName: TATA-box-binding protein / a.k.a: TBP, SPT15 / Number of Copies: 1 / Recombinant expression: Yes
MassTheoretical: 27 kDa
SourceSpecies: Saccharomyces cerevisiae (baker's yeast)
Source (engineered)Expression System: Escherichia coli (E. coli) / Vector: pOPINE / Strain: BL21(DE3)RIL

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Component #5: protein, Transcription Factor IIE

ProteinName: Transcription Factor IIE / a.k.a: TFIIETranscription factor II E / Number of Copies: 1 / Recombinant expression: Yes
MassTheoretical: 91 kDa
SourceSpecies: Saccharomyces cerevisiae (baker's yeast)
Source (engineered)Expression System: Escherichia coli (E. coli) / Vector: pET21 / Strain: BL21(DE3)RIL

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Component #6: protein, Transcription Factor IIF

ProteinName: Transcription Factor IIF / a.k.a: TFIIFTranscription factor II F / Recombinant expression: Yes / Number of Copies: 1
MassTheoretical: 129 kDa
SourceSpecies: Saccharomyces cerevisiae (baker's yeast)
Source (engineered)Expression System: Escherichia coli (E. coli) / Vector: pETDuet-1 / Strain: BL21(DE3)RIL

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Component #7: nucleic-acid, synthetic promoter DNA construct

nucleic acidName: synthetic promoter DNA construct / Class: DNA
Details: closed promoter DNA was opened by transcription machinery
Structure: DOUBLE HELIX / Synthetic: Yes
MassTheoretical: 44 kDa
SourceSpecies: unidentified (others)

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Experimental details

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Sample preparation

SpecimenSpecimen state: Particle / Method: cryo EM
Sample solutionSpecimen conc.: 0.3 mg/mL
Buffer solution: 25 mM HEPES-KOH pH 7.5, 150 mM potassium acetate, 2 mM MgCl2, 5 mM DTT
pH: 7.5
Support filmR3.5/1 holey carbon grids (Quantifoil)
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 %
Method: Quantifoil R 3.5/1 holey carbon grids were glow-discharged before deposition of 4.5 microliters of sample. Grids were then blotted for 8.5 s and plunge-frozen in liquid ethane.

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
ImagingMicroscope: FEI TITAN KRIOS / Date: Jun 20, 2016
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 40 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 37000 X (nominal), 37037 X (calibrated) / Imaging mode: BRIGHT FIELD / Defocus: 800 - 5000 nm / Energy filter: GIF Quantum / Energy window: 0-20 eV
Specimen HolderModel: FEI TITAN KRIOS AUTOGRID HOLDER
CameraDetector: GATAN K2 SUMMIT (4k x 4k)

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Image acquisition

Image acquisitionNumber of digital images: 959

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C1 (asymmetric) / Number of projections: 79797
3D reconstructionAlgorithm: RELION 1.3 / Software: RELION / CTF correction: Each particle / Resolution: 6.1 Å / Resolution method: FSC 0.143, gold-standard

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