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- EMDB-3381: Transcription initiation complex structures elucidate DNA opening... -

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Basic information

Entry
Database: EMDB / ID: EMD-3381
TitleTranscription initiation complex structures elucidate DNA opening (OC4-focused)
Map datasharpened OC4-focused map
Sample
  • Sample: yeast Pol II transcription initiation complex (open DNA)
  • Protein or peptide: DNA-directed RNA polymerase II
  • Protein or peptide: Transcription Factor IIA
  • Protein or peptide: Transcription Factor IIB
  • Protein or peptide: TATA-box-binding protein
  • Protein or peptide: Transcription Factor IIE
  • Protein or peptide: Transcription Factor IIF
  • DNA: synthetic promoter DNA construct (open)
KeywordsGene expression / Transcription initiation
Biological speciesSaccharomyces cerevisiae (brewer's yeast) / unidentified (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.09 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
DepositionMar 9, 2016-
Header (metadata) releaseApr 13, 2016-
Map releaseMay 18, 2016-
UpdateJun 15, 2016-
Current statusJun 15, 2016Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.05
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.05
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_3381.png

Downloads & links

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Map

FileDownload / File: emd_3381.map.gz / Format: CCP4 / Size: 100.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationsharpened OC4-focused map
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

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.05 / Movie #1: 0.05
Minimum - Maximum-0.11503202 - 0.19852653
Average (Standard dev.)-0.00011978 (±0.00388128)
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-0.1150.199-0.000

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Supplemental data

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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)
Components
  • Sample: yeast Pol II transcription initiation complex (open DNA)
  • Protein or peptide: DNA-directed RNA polymerase II
  • Protein or peptide: Transcription Factor IIA
  • Protein or peptide: Transcription Factor IIB
  • Protein or peptide: TATA-box-binding protein
  • Protein or peptide: Transcription Factor IIE
  • Protein or peptide: Transcription Factor IIF
  • DNA: synthetic promoter DNA construct (open)

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Supramolecule #1000: yeast Pol II transcription initiation complex (open DNA)

SupramoleculeName: yeast Pol II transcription initiation complex (open DNA)
type: sample / ID: 1000 / Oligomeric state: Monomer / Number unique components: 7
Molecular weightTheoretical: 890 KDa

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Macromolecule #1: DNA-directed RNA polymerase II

MacromoleculeName: DNA-directed RNA polymerase II / type: protein_or_peptide / ID: 1 / Name.synonym: RNA polymerase II / Number of copies: 1 / Recombinant expression: No
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / Strain: BJ5464 Rpb3 His-Bio / synonym: Baker's yeast
Molecular weightTheoretical: 513 KDa

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Macromolecule #2: Transcription Factor IIA

MacromoleculeName: Transcription Factor IIA / type: protein_or_peptide / ID: 2 / Name.synonym: TFIIA / Number of copies: 1 / Recombinant expression: Yes
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: Baker's yeast
Molecular weightTheoretical: 45 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: BL21(DE3)RIL / Recombinant plasmid: pOPINE

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Macromolecule #3: Transcription Factor IIB

MacromoleculeName: Transcription Factor IIB / type: protein_or_peptide / ID: 3 / Name.synonym: TFIIB / Number of copies: 1 / Recombinant expression: Yes
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: Baker's yeast
Molecular weightTheoretical: 38 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: BL21(DE3)RIL / Recombinant plasmid: pET21b

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Macromolecule #4: TATA-box-binding protein

MacromoleculeName: TATA-box-binding protein / type: protein_or_peptide / ID: 4 / Name.synonym: TBP, SPT15 / Number of copies: 1 / Recombinant expression: Yes
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: Baker's yeast
Molecular weightTheoretical: 27 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: BL21(DE3)RIL / Recombinant plasmid: pOPINE

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Macromolecule #5: Transcription Factor IIE

MacromoleculeName: Transcription Factor IIE / type: protein_or_peptide / ID: 5 / Name.synonym: TFIIE / Number of copies: 1 / Recombinant expression: Yes
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: Baker's yeast
Molecular weightTheoretical: 91 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: BL21(DE3)RIL / Recombinant plasmid: pET21

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Macromolecule #6: Transcription Factor IIF

MacromoleculeName: Transcription Factor IIF / type: protein_or_peptide / ID: 6 / Name.synonym: TFIIF / Number of copies: 1 / Recombinant expression: Yes
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: Baker's yeast
Molecular weightTheoretical: 129 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: BL21(DE3)RIL / Recombinant plasmid: pETDuet-1

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Macromolecule #7: synthetic promoter DNA construct (open)

MacromoleculeName: synthetic promoter DNA construct (open) / type: dna / ID: 7 / Details: Contains a 15 nucleotide mismatch bubble / Classification: DNA / Structure: DOUBLE HELIX / Synthetic?: Yes
Source (natural)Organism: unidentified (others)
Molecular weightTheoretical: 44 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

Concentration0.3 mg/mL
BufferpH: 7.5
Details: 25 mM HEPES-KOH pH 7.5, 150 mM potassium acetate, 2 mM MgCl2, 5 mM DTT
GridDetails: R3.5/1 holey carbon grids (Quantifoil)
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Instrument: FEI VITROBOT MARK IV
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

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Quantum / Energy filter - Lower energy threshold: 0.0 eV / Energy filter - Upper energy threshold: 20.0 eV
DateApr 26, 2016
Image recordingCategory: CCD / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Number real images: 1756 / Average electron dose: 33 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 37037 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.3 mm / Nominal defocus max: 4.2 µm / Nominal defocus min: 0.7 µm / Nominal magnification: 37000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: Each particle
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 4.09 Å / Resolution method: OTHER / Software - Name: RELION / Number images used: 109466

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