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- EMDB-3218: Structure of transcribing mammalian RNA polymerase II (EC1) -

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Basic information

Entry
Database: EMDB / ID: EMD-3218
TitleStructure of transcribing mammalian RNA polymerase II (EC1)
Map dataBovine Pol II elongation complex EC1, B-factor sharpened 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 / homology
Function and homology information


Formation of RNA Pol II elongation complex / Formation of the Early Elongation Complex / B-WICH complex positively regulates rRNA expression / Transcriptional regulation by small RNAs / TP53 Regulates Transcription of DNA Repair Genes / FGFR2 alternative splicing / RNA polymerase II transcribes snRNA genes / mRNA Capping / mRNA Splicing - Minor Pathway / RNA Polymerase I Transcription Initiation ...Formation of RNA Pol II elongation complex / Formation of the Early Elongation Complex / B-WICH complex positively regulates rRNA expression / Transcriptional regulation by small RNAs / TP53 Regulates Transcription of DNA Repair Genes / FGFR2 alternative splicing / RNA polymerase II transcribes snRNA genes / mRNA Capping / mRNA Splicing - Minor Pathway / RNA Polymerase I Transcription Initiation / RNA Polymerase I Promoter Escape / RNA Polymerase II Promoter Escape / RNA Polymerase II Transcription Pre-Initiation And Promoter Opening / RNA Polymerase I Transcription Termination / RNA Polymerase II Transcription Initiation / RNA Polymerase II Transcription Elongation / RNA Polymerase II Transcription Initiation And Promoter Clearance / RNA Polymerase III Transcription Initiation From Type 1 Promoter / RNA Polymerase III Transcription Initiation From Type 2 Promoter / RNA Polymerase III Transcription Initiation From Type 3 Promoter / RNA Pol II CTD phosphorylation and interaction with CE / Estrogen-dependent gene expression / RNA Polymerase II Pre-transcription Events / Processing of Capped Intron-Containing Pre-mRNA / : / mRNA Splicing - Major Pathway / Formation of TC-NER Pre-Incision Complex / Dual incision in TC-NER / Gap-filling DNA repair synthesis and ligation in TC-NER / : / : / positive regulation of nuclear-transcribed mRNA poly(A) tail shortening / maintenance of transcriptional fidelity during transcription elongation by RNA polymerase II / tRNA transcription by RNA polymerase III / RNA polymerase II activity / organelle membrane / positive regulation of translational initiation / transcription-coupled nucleotide-excision repair / transcription by RNA polymerase I / RNA polymerase I complex / RNA polymerase III complex / transcription by RNA polymerase III / RNA polymerase II, core complex / translation initiation factor binding / core promoter sequence-specific DNA binding / transcription initiation at RNA polymerase II promoter / protein-DNA complex / P-body / euchromatin / ribonucleoside binding / DNA-directed 5'-3' RNA polymerase activity / DNA-directed RNA polymerase / single-stranded DNA binding / single-stranded RNA binding / chromosome, telomeric region / transcription by RNA polymerase II / nucleic acid binding / protein dimerization activity / nuclear speck / nucleotide binding / chromatin binding / nucleolus / DNA binding / zinc ion binding / metal ion binding / nucleus / cytosol
Similarity search - Function
RNA-binding domain, S1 / DNA-directed RNA polymerase II subunit Rpb4-like / RNA polymerase Rpb4/RPC9, core / DNA-directed RNA-polymerase II subunit / RNA polymerase II, heptapeptide repeat, eukaryotic / RNA polymerase Rpb1, domain 6 / RNA polymerase Rpb1, domain 6 / RNA polymerase Rpb1 C-terminal repeat / Eukaryotic RNA polymerase II heptapeptide repeat. / RNA polymerase Rpb1, domain 7 ...RNA-binding domain, S1 / DNA-directed RNA polymerase II subunit Rpb4-like / RNA polymerase Rpb4/RPC9, core / DNA-directed RNA-polymerase II subunit / RNA polymerase II, heptapeptide repeat, eukaryotic / RNA polymerase Rpb1, domain 6 / RNA polymerase Rpb1, domain 6 / RNA polymerase Rpb1 C-terminal repeat / Eukaryotic RNA polymerase II heptapeptide repeat. / RNA polymerase Rpb1, domain 7 / RNA polymerase Rpb1, domain 7 superfamily / RNA polymerase Rpb1, domain 7 / Pol II subunit B9, C-terminal zinc ribbon / RNA polymerase RBP11 / Rpb4/RPC9 superfamily / RNA polymerase subunit Rpb4/RPC9 / RNA polymerase Rpb4 / Zinc finger TFIIS-type signature. / HRDC-like superfamily / RNA polymerase subunit Rpb7-like / RNA polymerase Rpb7-like , N-terminal / RNA polymerase Rpb7-like, N-terminal domain superfamily / SHS2 domain found in N terminus of Rpb7p/Rpc25p/MJ0397 / RNA polymerase Rpb2, domain 4 / RNA polymerase Rpb2, domain 4 / RNA polymerase Rpb2, domain 5 / RNA polymerase Rpb2, domain 5 / DNA-directed RNA polymerase subunit RPABC5/Rpb10 / RNA polymerases, subunit N, zinc binding site / RNA polymerase subunit RPB10 / RNA polymerases N / 8 kDa subunit / RNA polymerases N / 8 Kd subunits signature. / DNA-directed RNA polymerase, M/15kDa subunit / RNA polymerases M/15 Kd subunit / RNA polymerase subunit 9 / DNA-directed RNA polymerase M, 15kDa subunit, conserved site / RNA polymerases M / 15 Kd subunits signature. / DNA-directed RNA polymerase subunit/transcription factor S / RNA polymerase, Rpb8 / DNA-directed RNA polymerases I, II, and III subunit RPABC4 / RNA polymerase Rpb8 / RNA polymerase subunit 8 / RNA polymerase, Rpb5, N-terminal / RNA polymerase Rpb5, N-terminal domain superfamily / RNA polymerase Rpb5, N-terminal domain / DNA-directed RNA polymerase, subunit RPB6 / DNA directed RNA polymerase, 7 kDa subunit / RNA polymerase archaeal subunit P/eukaryotic subunit RPABC4 / RNA polymerase, subunit H/Rpb5, conserved site / RNA polymerases H / 23 Kd subunits signature. / RNA polymerase subunit CX / DNA-directed RNA polymerase Rpb11, 13-16kDa subunit, conserved site / RNA polymerases L / 13 to 16 Kd subunits signature. / DNA-directed RNA polymerase, 30-40kDa subunit, conserved site / DNA-directed RNA polymerase subunit Rpo3/Rpb3/RPAC1 / RNA polymerases D / 30 to 40 Kd subunits signature. / DNA-directed RNA polymerase, RBP11-like dimerisation domain / DNA-directed RNA polymerase subunit Rpo5/Rpb5 / RNA polymerase Rpb3/Rpb11 dimerisation domain / RNA polymerase, subunit H/Rpb5 C-terminal / RNA polymerase subunit RPABC4/transcription elongation factor Spt4 / RPB5-like RNA polymerase subunit superfamily / RNA polymerase Rpb5, C-terminal domain / Archaeal Rpo6/eukaryotic RPB6 RNA polymerase subunit / DNA-directed RNA polymerase, 14-18kDa subunit, conserved site / RNA polymerases K / 14 to 18 Kd subunits signature. / Zinc finger, TFIIS-type / Transcription factor S-II (TFIIS) / Zinc finger TFIIS-type profile. / C2C2 Zinc finger / Ribosomal protein S1-like RNA-binding domain / S1 RNA binding domain / S1 domain / RNA polymerase Rpb6 / RNA polymerase, subunit omega/Rpo6/RPB6 / RNA polymerase Rpb6 / RNA polymerase Rpb1, domain 3 superfamily / RNA polymerase Rpb1, clamp domain superfamily / RPB6/omega subunit-like superfamily / DNA-directed RNA polymerase, subunit beta-prime / RNA polymerase Rpb1, domain 3 / RNA polymerase Rpb1, domain 3 / RNA polymerase Rpb2, domain 2 superfamily / RNA polymerase Rpb1, domain 1 / RNA polymerase Rpb1, domain 1 / RNA polymerase, alpha subunit / RNA polymerase Rpb1, domain 4 / RNA polymerase Rpb1, domain 2 / RNA polymerase Rpb1, domain 4 / RNA polymerase, N-terminal / RNA polymerase Rpb1, funnel domain superfamily / RNA polymerase I subunit A N-terminus / RNA polymerase Rpb1, domain 5 / RNA polymerase Rpb1, domain 5 / RNA polymerase, beta subunit, protrusion / RNA polymerase beta subunit / DNA-directed RNA polymerase, insert domain / DNA-directed RNA polymerase, RpoA/D/Rpb3-type / RNA polymerase Rpb3/RpoA insert domain / RNA polymerase Rpb3/Rpb11 dimerisation domain
Similarity search - Domain/homology
DNA-directed RNA polymerase subunit beta / DNA-directed RNA polymerases I, II, and III subunit RPABC3 / DNA-directed RNA polymerase subunit / Polymerase (RNA) II (DNA directed) polypeptide D / DNA-directed RNA polymerases I, II, and III subunit RPABC1 / DNA-directed RNA polymerase II subunit RPB9 / DNA-directed RNA polymerases I, II, and III subunit RPABC5 / DNA-directed RNA polymerase II subunit RPB11 / DNA-directed RNA polymerases I, II, and III subunit RPABC2 / DNA-directed RNA polymerase II subunit RPB3 ...DNA-directed RNA polymerase subunit beta / DNA-directed RNA polymerases I, II, and III subunit RPABC3 / DNA-directed RNA polymerase subunit / Polymerase (RNA) II (DNA directed) polypeptide D / DNA-directed RNA polymerases I, II, and III subunit RPABC1 / DNA-directed RNA polymerase II subunit RPB9 / DNA-directed RNA polymerases I, II, and III subunit RPABC5 / DNA-directed RNA polymerase II subunit RPB11 / DNA-directed RNA polymerases I, II, and III subunit RPABC2 / DNA-directed RNA polymerase II subunit RPB3 / DNA-directed RNA polymerases I, II, and III subunit RPABC4 / DNA-directed RNA polymerase II subunit RPB7
Similarity search - Component
Biological speciesBos taurus (cattle) / Homo sapiens (human) / unidentified (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.4 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 20, 2016-
UpdateFeb 3, 2016-
Current statusFeb 3, 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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  • Surface view with fitted model
  • Atomic models: PDB-5flm
  • Surface level: 0.05
  • 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_3218.map.gz / Format: CCP4 / Size: 31.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationBovine Pol II elongation complex EC1, B-factor sharpened map
Voxel sizeX=Y=Z: 1.35 Å
Density
Contour LevelBy AUTHOR: 0.07 / Movie #1: 0.05
Minimum - Maximum-0.30116436 - 0.4999862
Average (Standard dev.)0.00011944 (±0.0182527)
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
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS204204204
D min/max/mean-0.3010.5000.000

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Supplemental data

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Supplemental map: EMD-3218 unsharp.map

FileEMD-3218_unsharp.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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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: 3.4 Å / Resolution method: OTHER / Software - Name: RELION / Number images used: 264134

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