|Entry||Database: EMDB / ID: EMD-10019|
|Title||RNA Polymerase I Open Complex conformation 2 focused refinement on CF|
|Sample||RNA Polymerase I Open Complex conformation 2 focused refinement on CF:|
RNA Polymerase I / DNA / transcription initiation factors
|Biological species||Saccharomyces cerevisiae (baker's yeast) / synthetic construct (others)|
|Method||single particle reconstruction / cryo EM / Resolution: 3 Å|
|Authors||Mueller CW / Sadian Y|
|Funding support||1 items |
|Citation||Journal: Nat Commun / Year: 2019|
Title: Molecular insight into RNA polymerase I promoter recognition and promoter melting.
Authors: Yashar Sadian / Florence Baudin / Lucas Tafur / Brice Murciano / Rene Wetzel / Felix Weis / Christoph W Müller /
Abstract: RNA polymerase I (Pol I) assembles with core factor (CF) and Rrn3 on the rDNA core promoter for transcription initiation. Here, we report cryo-EM structures of closed, intermediate and open Pol I ...RNA polymerase I (Pol I) assembles with core factor (CF) and Rrn3 on the rDNA core promoter for transcription initiation. Here, we report cryo-EM structures of closed, intermediate and open Pol I initiation complexes from 2.7 to 3.7 Å resolution to visualize Pol I promoter melting and to structurally and biochemically characterize the recognition mechanism of Pol I promoter DNA. In the closed complex, double-stranded DNA runs outside the DNA-binding cleft. Rotation of CF and upstream DNA with respect to Pol I and Rrn3 results in the spontaneous loading and opening of the promoter followed by cleft closure and positioning of the Pol I A49 tandem winged helix domain (tWH) onto DNA. Conformational rearrangement of A49 tWH leads to a clash with Rrn3 to initiate complex disassembly and promoter escape. Comprehensive insight into the Pol I transcription initiation cycle allows comparisons with promoter opening by Pol II and Pol III.
|Structure viewer||EM map: |
Downloads & links
|File||Download / File: emd_10019.map.gz / Format: CCP4 / Size: 91.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)|
|Projections & slices|
Images are generated by Spider.
|Voxel size||X=Y=Z: 1.32 Å|
|Symmetry||Space group: 1|
CCP4 map header:
-Entire RNA Polymerase I Open Complex conformation 2 focused refinement on CF
|Entire||Name: RNA Polymerase I Open Complex conformation 2 focused refinement on CF|
Number of components: 4
-Component #1: protein, RNA Polymerase I Open Complex conformation 2 focused ref...
|Protein||Name: RNA Polymerase I Open Complex conformation 2 focused refinement on CF|
Recombinant expression: No
-Component #2: protein, RNA Polymerase I
|Protein||Name: RNA Polymerase I / Recombinant expression: No|
|Source||Species: Saccharomyces cerevisiae (baker's yeast)|
-Component #3: protein, DNA
|Protein||Name: DNA / Recombinant expression: No|
|Source||Species: synthetic construct (others)|
|Source (engineered)||Expression System: synthetic construct (others)|
-Component #4: protein, transcription initiation factors
|Protein||Name: transcription initiation factors / Recombinant expression: No|
|Source||Species: Saccharomyces cerevisiae (baker's yeast)|
|Source (engineered)||Expression System: Escherichia coli (E. coli)|
|Specimen||Specimen state: Particle / Method: cryo EM|
|Sample solution||pH: 7.5|
|Vitrification||Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Temperature: 4 K / Humidity: 100 %|
-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: 1.57175 e/Å2 / Illumination mode: FLOOD BEAM|
|Lens||Imaging mode: BRIGHT FIELD|
|Specimen Holder||Model: OTHER|
|Camera||Detector: GATAN K2 SUMMIT (4k x 4k)|
|Processing||Method: single particle reconstruction / Number of projections: 59963|
|3D reconstruction||Resolution: 3 Å / Resolution method: FSC 0.143 CUT-OFF|
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