|Entry||Database: EMDB / ID: EMD-10489|
|Title||Structure of complete, activated transcription complex Pol II-DSIF-PAF-SPT6 uncovers allosteric elongation activation by RTF1 (Map 10)|
|Biological species||Homo sapiens (human)|
|Method||single particle reconstruction / cryo EM / Resolution: 3.7 Å|
|Authors||Vos SM / Farnung L / Cramer P|
|Funding support|| Germany, 4 items |
|Citation||Journal: Nat Struct Mol Biol / Year: 2020|
Title: Structure of complete Pol II-DSIF-PAF-SPT6 transcription complex reveals RTF1 allosteric activation.
Authors: Seychelle M Vos / Lucas Farnung / Andreas Linden / Henning Urlaub / Patrick Cramer /
Abstract: Transcription by RNA polymerase II (Pol II) is carried out by an elongation complex. We previously reported an activated porcine Pol II elongation complex, EC*, encompassing the human elongation ...Transcription by RNA polymerase II (Pol II) is carried out by an elongation complex. We previously reported an activated porcine Pol II elongation complex, EC*, encompassing the human elongation factors DSIF, PAF1 complex (PAF) and SPT6. Here we report the cryo-EM structure of the complete EC* that contains RTF1, a dissociable PAF subunit critical for chromatin transcription. The RTF1 Plus3 domain associates with Pol II subunit RPB12 and the phosphorylated C-terminal region of DSIF subunit SPT5. RTF1 also forms four α-helices that extend from the Plus3 domain along the Pol II protrusion and RPB10 to the polymerase funnel. The C-terminal 'fastener' helix retains PAF and is followed by a 'latch' that reaches the end of the bridge helix, a flexible element of the Pol II active site. RTF1 strongly stimulates Pol II elongation, and this requires the latch, possibly suggesting that RTF1 activates transcription allosterically by influencing Pol II translocation.
|Structure viewer||EM map: |
Downloads & links
|File||Download / File: emd_10489.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)|
|Projections & slices|
Images are generated by Spider.
|Voxel size||X=Y=Z: 1.049 Å|
|Symmetry||Space group: 1|
CCP4 map header:
-Half map: Half map 1
|Annotation||Half map 1|
|Projections & Slices|
-Half map: Half map 2
-Entire Complete EC*
|Entire||Name: Complete EC* / Number of components: 1|
-Component #1: protein, Complete EC*
|Protein||Name: Complete EC* / Recombinant expression: No|
|Mass||Theoretical: 1.34 MDa|
|Source||Species: Homo sapiens (human)|
|Specimen||Specimen state: Particle / Method: cryo EM|
|Sample solution||pH: 7.4|
|Vitrification||Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Temperature: 277 K / Humidity: 100 %|
Details: 2 microliters applied to both sides of grid. Sample incubated on grid for 10s prior to blotting. Blotting for 8.5s..
-Electron microscopy imaging
Model: Titan Krios / Image courtesy: FEI Company
|Imaging||Microscope: FEI TITAN KRIOS|
|Electron gun||Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 40 e/Å2 / Illumination mode: SPOT SCAN|
|Lens||Magnification: 130000 X (nominal) / Imaging mode: BRIGHT FIELD / Energy filter: GIF Bioquantum|
|Specimen Holder||Model: FEI TITAN KRIOS AUTOGRID HOLDER|
|Camera||Detector: GATAN K2 QUANTUM (4k x 4k)|
|Image acquisition||Number of digital images: 13679|
|Processing||Method: single particle reconstruction / Applied symmetry: C1 (asymmetric) / Number of projections: 35713|
|3D reconstruction||Software: RELION / Resolution: 3.7 Å / Resolution method: FSC 0.143 CUT-OFF|
|FSC plot (resolution estimation)|
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