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- EMDB-3220: Structure of transcribing mammalian RNA polymerase II (EC3) -

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Basic information

Entry
Database: EMDB / ID: EMD-3220
TitleStructure of transcribing mammalian RNA polymerase II (EC3)
Map dataBovine Pol II elongation complex EC3, unsharpened map
Sample
  • Sample: bovine Pol II elongation complex
  • Protein or peptide: DNA-directed RNA polymerase IIPolymerase
  • Protein or peptide: DNA-directed RNA polymerase II subunit GRINL1APolymerase
  • Other: DNA-RNA synthetic construct
Keywordstranscription / elongation
Function / homologyDNA-directed 5'-3' RNA polymerase activity
Function and homology information
Biological speciesBos taurus (cattle) / Homo sapiens (human) / unidentified (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.7 Å
AuthorsBernecky C / Herzog F / Baumeister W / Plitzko JM / Cramer P
CitationJournal: Nature / Year: 2016
Title: Structure of transcribing mammalian RNA polymerase II.
Authors: Carrie Bernecky / Franz Herzog / Wolfgang Baumeister / Jürgen M Plitzko / Patrick Cramer /
Abstract: RNA polymerase (Pol) II produces messenger RNA during transcription of protein-coding genes in all eukaryotic cells. The Pol II structure is known at high resolution from X-ray crystallography for ...RNA polymerase (Pol) II produces messenger RNA during transcription of protein-coding genes in all eukaryotic cells. The Pol II structure is known at high resolution from X-ray crystallography for two yeast species. Structural studies of mammalian Pol II, however, remain limited to low-resolution electron microscopy analysis of human Pol II and its complexes with various proteins. Here we report the 3.4 Å resolution cryo-electron microscopy structure of mammalian Pol II in the form of a transcribing complex comprising DNA template and RNA transcript. We use bovine Pol II, which is identical to the human enzyme except for seven amino-acid residues. The obtained atomic model closely resembles its yeast counterpart, but also reveals unknown features. Binding of nucleic acids to the polymerase involves 'induced fit' of the mobile Pol II clamp and active centre region. DNA downstream of the transcription bubble contacts a conserved 'TPSA motif' in the jaw domain of the Pol II subunit RPB5, an interaction that is apparently already established during transcription initiation. Upstream DNA emanates from the active centre cleft at an angle of approximately 105° with respect to downstream DNA. This position of upstream DNA allows for binding of the general transcription elongation factor DSIF (SPT4-SPT5) that we localize over the active centre cleft in a conserved position on the clamp domain of Pol II. Our results define the structure of mammalian Pol II in its functional state, indicate that previous crystallographic analysis of yeast Pol II is relevant for understanding gene transcription in all eukaryotes, and provide a starting point for a mechanistic analysis of human transcription.
History
DepositionOct 26, 2015-
Header (metadata) releaseNov 11, 2015-
Map releaseJan 27, 2016-
UpdateFeb 17, 2016-
Current statusFeb 17, 2016Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.024
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.024
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_3220.tif

Downloads & links

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Map

FileDownload / File: emd_3220.map.gz / Format: CCP4 / Size: 31.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationBovine Pol II elongation complex EC3, unsharpened map
Voxel sizeX=Y=Z: 1.35 Å
Density
Contour LevelBy AUTHOR: 0.024 / Movie #1: 0.024
Minimum - Maximum-0.03549683 - 0.10071836
Average (Standard dev.)-0.00005501 (±0.0055686)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions204204204
Spacing204204204
CellA=B=C: 275.4 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.351.351.35
M x/y/z204204204
origin x/y/z0.0000.0000.000
length x/y/z275.400275.400275.400
α/β/γ90.00090.00090.000
start NX/NY/NZ-180-180-179
NX/NY/NZ360360360
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS204204204
D min/max/mean-0.0350.101-0.000

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Supplemental data

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Supplemental map: EMD-3220 autob.map

FileEMD-3220_autob.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 : bovine Pol II elongation complex

EntireName: bovine Pol II elongation complex
Components
  • Sample: bovine Pol II elongation complex
  • Protein or peptide: DNA-directed RNA polymerase IIPolymerase
  • Protein or peptide: DNA-directed RNA polymerase II subunit GRINL1APolymerase
  • Other: DNA-RNA synthetic construct

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Supramolecule #1000: bovine Pol II elongation complex

SupramoleculeName: bovine Pol II elongation complex / type: sample / ID: 1000
Details: Recombinant human Gdown1 was present during sample preparation but density was not observed in this map.
Number unique components: 3
Molecular weightTheoretical: 590 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 / Oligomeric state: Monomer / Recombinant expression: No
Source (natural)Organism: Bos taurus (cattle) / synonym: Cow / Tissue: Thymus
Molecular weightTheoretical: 520 KDa
SequenceGO: DNA-directed 5'-3' RNA polymerase activity

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Macromolecule #2: DNA-directed RNA polymerase II subunit GRINL1A

MacromoleculeName: DNA-directed RNA polymerase II subunit GRINL1A / type: protein_or_peptide / ID: 2 / Name.synonym: Gdown1 / Number of copies: 1 / Oligomeric state: Monomer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human
Molecular weightTheoretical: 40 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: BL21(DE3)RIL / Recombinant plasmid: pOPINB

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Macromolecule #3: DNA-RNA synthetic construct

MacromoleculeName: DNA-RNA synthetic construct / type: other / ID: 3 / Name.synonym: DNA-RNA elongation scaffold / Classification: DNA/RNA / Structure: OTHER / Synthetic?: Yes
Source (natural)Organism: unidentified (others)
Molecular weightTheoretical: 30 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: 7.25 / Details: 150 mM NaCl, 5 mM HEPES, 0.01 mM ZnCl2, 10 mM DTT
GridDetails: Quantifoil R 3.5/1 holey carbon grids
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Instrument: FEI VITROBOT MARK IV
Method: Four microliters of sample was applied to glow-discharged Quantifoil R 3.5/1 holey carbon grids, which were then blotted for 8.5s and plunge-frozen in liquid ethane.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 37037 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2 mm / Nominal defocus max: 3.1 µm / Nominal defocus min: 0.6 µm / Nominal magnification: 37000
Specialist opticsEnergy filter - Name: GIF Quantum / Energy filter - Lower energy threshold: 0.0 eV / Energy filter - Upper energy threshold: 20.0 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
DateDec 1, 2014
Image recordingCategory: CCD / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Number real images: 1172 / Average electron dose: 43 e/Å2
Details: Each movie image was collected over 8 s fractionated into 40 frames (0.2 s each).
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: 3.7 Å / Resolution method: OTHER / Software - Name: RELION / Number images used: 184122

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