|Entry||Database: EMDB / ID: EMD-4149|
|Title||Structure of RNA polymerase I transcribing rDNA genes|
|Sample||RNA polymerase I|
|Biological species||Saccharomyces cerevisiae (baker's yeast)|
|Method||subtomogram averaging / cryo EM / Resolution: 29 Å|
|Authors||Neyer S / Kunz M / Geiss C / Hantsche M / Hodirnau VV / Seybert A / Engel C / Scheffer MP / Cramer P / Frangakis AS|
|Citation||Journal: Nature / Year: 2016|
Title: Structure of RNA polymerase I transcribing ribosomal DNA genes.
Authors: Simon Neyer / Michael Kunz / Christian Geiss / Merle Hantsche / Victor-Valentin Hodirnau / Anja Seybert / Christoph Engel / Margot P Scheffer / Patrick Cramer / Achilleas S Frangakis /
Abstract: RNA polymerase I (Pol I) is a highly processive enzyme that transcribes ribosomal DNA (rDNA) and regulates growth of eukaryotic cells. Crystal structures of free Pol I from the yeast Saccharomyces ...RNA polymerase I (Pol I) is a highly processive enzyme that transcribes ribosomal DNA (rDNA) and regulates growth of eukaryotic cells. Crystal structures of free Pol I from the yeast Saccharomyces cerevisiae have revealed dimers of the enzyme stabilized by a 'connector' element and an expanded cleft containing the active centre in an inactive conformation. The central bridge helix was unfolded and a Pol-I-specific 'expander' element occupied the DNA-template-binding site. The structure of Pol I in its active transcribing conformation has yet to be determined, whereas structures of Pol II and Pol III have been solved with bound DNA template and RNA transcript. Here we report structures of active transcribing Pol I from yeast solved by two different cryo-electron microscopy approaches. A single-particle structure at 3.8 Å resolution reveals a contracted active centre cleft with bound DNA and RNA, and a narrowed pore beneath the active site that no longer holds the RNA-cleavage-stimulating domain of subunit A12.2. A structure at 29 Å resolution that was determined from cryo-electron tomograms of Pol I enzymes transcribing cellular rDNA confirms contraction of the cleft and reveals that incoming and exiting rDNA enclose an angle of around 150°. The structures suggest a model for the regulation of transcription elongation in which contracted and expanded polymerase conformations are associated with active and inactive states, respectively.
|Structure viewer||EM map: |
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|File||Download / File: emd_4149.map.gz / Format: CCP4 / Size: 1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)|
|Projections & slices|
Images are generated by Spider.
|Voxel size||X=Y=Z: 4 Å|
|Symmetry||Space group: 1|
CCP4 map header:
-Entire RNA polymerase I
|Entire||Name: RNA polymerase I / Number of components: 1|
-Component #1: protein, RNA polymerase I
|Protein||Name: RNA polymerase I / Recombinant expression: No|
|Source||Species: Saccharomyces cerevisiae (baker's yeast)|
|Specimen||Specimen state: Cell / Method: cryo EM|
|Sample solution||pH: 9|
|Vitrification||Cryogen name: ETHANE|
-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: 1.2 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: subtomogram averaging / Number of subtomograms: 225|
|3D reconstruction||Resolution: 29 Å / Resolution method: FSC 0.5 CUT-OFF|
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