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Yorodumi- EMDB-5443: Regulation of mammalian transcription by Gdown1 through a novel s... -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-5443 | |||||||||
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Title | Regulation of mammalian transcription by Gdown1 through a novel steric crosstalk revealed by cryo-EM | |||||||||
Map data | Reconstruction of Bovine RNA polymerase II elongation complex with Gdown1 | |||||||||
Sample |
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Keywords | RNA polymerase / Mediator / Gdown1 / TFIIF | |||||||||
Biological species | Bos taurus (cattle) | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 19.0 Å | |||||||||
Authors | Wu YM / Chang JW / Wang CH / Lin YC / Wu PL / Huang SH / Chang CC / Hu X / Gnatt A / Chang WH | |||||||||
Citation | Journal: EMBO J / Year: 2012 Title: Regulation of mammalian transcription by Gdown1 through a novel steric crosstalk revealed by cryo-EM. Authors: Yi-Min Wu / Jen-Wei Chang / Chun-Hsiung Wang / Yen-Chen Lin / Pei-lun Wu / Shih-hsin Huang / Chia-Chi Chang / Xiaopeng Hu / Averell Gnatt / Wei-hau Chang / Abstract: In mammals, a distinct RNA polymerase II form, RNAPII(G) contains a novel subunit Gdown1 (encoded by POLR2M), which represses gene activation, only to be reversed by the multisubunit Mediator co- ...In mammals, a distinct RNA polymerase II form, RNAPII(G) contains a novel subunit Gdown1 (encoded by POLR2M), which represses gene activation, only to be reversed by the multisubunit Mediator co-activator. Here, we employed single-particle cryo-electron microscopy (cryo-EM) to disclose the architectures of RNAPII(G), RNAPII and RNAPII in complex with the transcription initiation factor TFIIF, all to ~19 Å. Difference analysis mapped Gdown1 mostly to the RNAPII Rpb5 shelf-Rpb1 jaw, supported by antibody labelling experiments. These structural features correlate with the moderate increase in the efficiency of RNA chain elongation by RNAP II(G). In addition, our updated RNAPII-TFIIF map showed that TFIIF tethers multiple regions surrounding the DNA-binding cleft, in agreement with cross-linking and biochemical mapping. Gdown1's binding sites overlap extensively with those of TFIIF, with Gdown1 sterically excluding TFIIF from RNAPII, herein demonstrated by competition assays using size exclusion chromatography. In summary, our work establishes a structural basis for Gdown1 impeding initiation at promoters, by obstruction of TFIIF, accounting for an additional dependent role of Mediator in activated 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_5443.map.gz | 12 MB | EMDB map data format | |
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Header (meta data) | emd-5443-v30.xml emd-5443.xml | 8.8 KB 8.8 KB | Display Display | EMDB header |
Images | emd_5443_1.jpg | 62.7 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-5443 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-5443 | HTTPS FTP |
-Validation report
Summary document | emd_5443_validation.pdf.gz | 78.5 KB | Display | EMDB validaton report |
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Full document | emd_5443_full_validation.pdf.gz | 77.6 KB | Display | |
Data in XML | emd_5443_validation.xml.gz | 494 B | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-5443 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-5443 | HTTPS FTP |
-Related structure data
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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-Map
File | Download / File: emd_5443.map.gz / Format: CCP4 / Size: 12.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | Reconstruction of Bovine RNA polymerase II elongation complex with Gdown1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 1.9 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Sample components
-Entire : Bovine RNA polymerase II elongation complex with Gdown1
Entire | Name: Bovine RNA polymerase II elongation complex with Gdown1 |
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Components |
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-Supramolecule #1000: Bovine RNA polymerase II elongation complex with Gdown1
Supramolecule | Name: Bovine RNA polymerase II elongation complex with Gdown1 type: sample / ID: 1000 / Number unique components: 1 |
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Molecular weight | Theoretical: 560 KDa |
-Macromolecule #1: RNA polymerase II elongation complex
Macromolecule | Name: RNA polymerase II elongation complex / type: protein_or_peptide / ID: 1 / Oligomeric state: monomer / Recombinant expression: No / Database: NCBI |
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Source (natural) | Organism: Bos taurus (cattle) / synonym: bovine / Tissue: thymus |
Molecular weight | Theoretical: 560 KDa |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 1 mg/mL |
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Buffer | pH: 8 / Details: 50 mM Tris-Cl, 200 mM KOAc, 5mM MgCl2, 5 mM DDT |
Grid | Details: 200 mesh quantifoil grid with thin carbon layer |
Vitrification | Cryogen name: ETHANE / Chamber humidity: 80 % / Instrument: HOMEMADE PLUNGER |
-Electron microscopy
Microscope | JEOL 2010F |
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Date | May 8, 2012 |
Image recording | Digitization - Scanner: ZEISS SCAI / Number real images: 150 / Average electron dose: 20 e/Å2 / Bits/pixel: 16 |
Electron beam | Acceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Illumination mode: SPOT SCAN / Imaging mode: DARK FIELD |
Sample stage | Specimen holder model: GATAN LIQUID NITROGEN |
-Image processing
CTF correction | Details: Each particle |
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Final reconstruction | Resolution.type: BY AUTHOR / Resolution: 19.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: Spider, EMAN2 / Number images used: 24000 |
Final two d classification | Number classes: 128 |