+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-3218 | |||||||||
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Title | Structure of transcribing mammalian RNA polymerase II (EC1) | |||||||||
Map data | Bovine Pol II elongation complex EC1, B-factor sharpened map | |||||||||
Sample |
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Keywords | transcription / elongation | |||||||||
Function / homology | Function and homology information Formation of RNA Pol II elongation complex / Formation of the Early Elongation Complex / 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 ...Formation of RNA Pol II elongation complex / Formation of the Early Elongation Complex / 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 / B-WICH complex positively regulates rRNA expression / 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 / DNA/RNA hybrid binding / maintenance of transcriptional fidelity during transcription elongation by RNA polymerase II / transcription by RNA polymerase I / positive regulation of nuclear-transcribed mRNA poly(A) tail shortening / nuclear-transcribed mRNA catabolic process / transcription by RNA polymerase III / RNA polymerase II activity / transcription-coupled nucleotide-excision repair / transcription elongation by RNA polymerase I / tRNA transcription by RNA polymerase III / RNA polymerase I complex / RNA polymerase III complex / positive regulation of translational initiation / RNA polymerase II, core complex / core promoter sequence-specific DNA binding / translation initiation factor binding / protein-DNA complex / transcription initiation at RNA polymerase II promoter / P-body / euchromatin / ribonucleoside binding / DNA-directed 5'-3' RNA polymerase activity / DNA-directed RNA polymerase / single-stranded DNA binding / Hydrolases; Acting on ester bonds; Exoribonucleases producing 5'-phosphomonoesters / transcription by RNA polymerase II / nucleic acid binding / chromosome, telomeric region / single-stranded RNA binding / protein dimerization activity / hydrolase activity / RNA-directed RNA polymerase / RNA-dependent RNA polymerase activity / nucleotide binding / chromatin binding / nucleolus / DNA binding / zinc ion binding / nucleus / metal ion binding / cytosol Similarity search - Function | |||||||||
Biological species | Bos taurus (cattle) / Homo sapiens (human) / unidentified (others) | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 3.4 Å | |||||||||
Authors | Bernecky C / Herzog F / Baumeister W / Plitzko JM / Cramer P | |||||||||
Citation | Journal: 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 |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | EM map: SurfViewMolmilJmol/JSmol |
Supplemental images |
-Downloads & links
-EMDB archive
Map data | emd_3218.map.gz | 30.3 MB | EMDB map data format | |
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Header (meta data) | emd-3218-v30.xml emd-3218.xml | 10.8 KB 10.8 KB | Display Display | EMDB header |
Images | emd_3218.tif | 150.7 KB | ||
Others | emd_3218_additional_1.map.gz | 30.1 MB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-3218 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-3218 | HTTPS FTP |
-Validation report
Summary document | emd_3218_validation.pdf.gz | 289 KB | Display | EMDB validaton report |
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Full document | emd_3218_full_validation.pdf.gz | 288.1 KB | Display | |
Data in XML | emd_3218_validation.xml.gz | 5.7 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-3218 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-3218 | HTTPS FTP |
-Related structure data
Related structure data | 5flmMC 3219C 3220C M: atomic model generated by this map C: citing same article (ref.) |
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Similar structure data |
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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Related items in Molecule of the Month |
-Map
File | Download / File: emd_3218.map.gz / Format: CCP4 / Size: 31.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | Bovine Pol II elongation complex EC1, B-factor sharpened map | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Projections & slices | Image control
Images are generated by Spider. | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 1.35 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Supplemental map: emd 3218 additional 1.map
File | emd_3218_additional_1.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Sample components
-Entire : bovine Pol II elongation complex
Entire | Name: bovine Pol II elongation complex |
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Components |
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-Supramolecule #1000: bovine Pol II elongation complex
Supramolecule | Name: 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 |
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Molecular weight | Theoretical: 590 KDa |
-Macromolecule #1: DNA-directed RNA polymerase II
Macromolecule | Name: 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 |
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Source (natural) | Organism: Bos taurus (cattle) / synonym: Cow / Tissue: Thymus |
Molecular weight | Theoretical: 520 KDa |
Sequence | GO: DNA-directed 5'-3' RNA polymerase activity |
-Macromolecule #2: DNA-directed RNA polymerase II subunit GRINL1A
Macromolecule | Name: 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 |
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Source (natural) | Organism: Homo sapiens (human) / synonym: Human |
Molecular weight | Theoretical: 40 KDa |
Recombinant expression | Organism: Escherichia coli (E. coli) / Recombinant strain: BL21(DE3)RIL / Recombinant plasmid: pOPINB |
-Macromolecule #3: DNA-RNA synthetic construct
Macromolecule | Name: DNA-RNA synthetic construct / type: other / ID: 3 / Name.synonym: DNA-RNA elongation scaffold / Classification: DNA/RNA / Structure: OTHER / Synthetic?: Yes |
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Source (natural) | Organism: unidentified (others) |
Molecular weight | Theoretical: 30 KDa |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 0.3 mg/mL |
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Buffer | pH: 7.25 / Details: 150 mM NaCl, 5 mM HEPES, 0.01 mM ZnCl2, 10 mM DTT |
Grid | Details: Quantifoil R 3.5/1 holey carbon grids |
Vitrification | Cryogen 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. |
-Electron microscopy
Microscope | FEI TITAN KRIOS |
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Specialist optics | Energy filter - Name: GIF Quantum / Energy filter - Lower energy threshold: 0.0 eV / Energy filter - Upper energy threshold: 20.0 eV |
Date | Dec 1, 2014 |
Image recording | Category: 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). |
Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Calibrated magnification: 37037 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2 mm / Nominal defocus max: 3.1 µm / Nominal defocus min: 0.6 µm / Nominal magnification: 37000 |
Sample stage | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
-Image processing
CTF correction | Details: Each particle |
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Final reconstruction | Applied 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 |