|Entry||Database: EMDB / ID: EMD-1754|
|Title||YEAST RNA POLYMERASE III-DNA-RNA COMPLEX|
|Sample||RNA polymerase III-DNA-RNA complex:|
RNA polymerase III / nucleic-acidNucleic acid
|Keywords||Transcription / RNA polymerase / tRNA|
|Biological species||Saccharomyces cerevisiae (baker's yeast) / synthetic construct (others)|
|Method||single particle reconstruction / cryo EM / Resolution: 19 Å|
|Authors||Vannini A / Ringel R / Kusser AG / Berninghausen O / Kassavetis GA / Cramer P|
|Citation||Journal: Cell / Year: 2010|
Title: Molecular basis of RNA polymerase III transcription repression by Maf1.
Authors: Alessandro Vannini / Rieke Ringel / Anselm G Kusser / Otto Berninghausen / George A Kassavetis / Patrick Cramer /
Abstract: RNA polymerase III (Pol III) transcribes short RNAs required for cell growth. Under stress conditions, the conserved protein Maf1 rapidly represses Pol III transcription. We report the crystal ...RNA polymerase III (Pol III) transcribes short RNAs required for cell growth. Under stress conditions, the conserved protein Maf1 rapidly represses Pol III transcription. We report the crystal structure of Maf1 and cryo-electron microscopic structures of Pol III, an active Pol III-DNA-RNA complex, and a repressive Pol III-Maf1 complex. Binding of DNA and RNA causes ordering of the Pol III-specific subcomplex C82/34/31 that is required for transcription initiation. Maf1 binds the Pol III clamp and rearranges C82/34/31 at the rim of the active center cleft. This impairs recruitment of Pol III to a complex of promoter DNA with the initiation factors Brf1 and TBP and thus prevents closed complex formation. Maf1 does however not impair binding of a DNA-RNA scaffold and RNA synthesis. These results explain how Maf1 specifically represses transcription initiation from Pol III promoters and indicate that Maf1 also prevents reinitiation by binding Pol III during transcription elongation.
|Structure viewer||EM map: |
Downloads & links
|File||Download / File: emd_1754.map.gz / Format: CCP4 / Size: 2.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)|
|Projections & slices|
Images are generated by Spider.
|Voxel size||X=Y=Z: 3.31 Å|
|Symmetry||Space group: 1|
CCP4 map header:
-Entire RNA polymerase III-DNA-RNA complex
|Entire||Name: RNA polymerase III-DNA-RNA complex / Details: The sample was monodisperse / Number of components: 2 / Oligomeric State: Dimeric|
|Mass||Theoretical: 700 kDa / Experimental: 700 kDa / Measured by: Native Mass Spectrometry|
-Component #1: protein, RNA polymerase III
|Protein||Name: RNA polymerase III / a.k.a: RNA polymerase III / Oligomeric Details: Monomer / Number of Copies: 1 / Recombinant expression: Yes|
|Mass||Theoretical: 700 kDa / Experimental: 700 kDa|
|Source||Species: Saccharomyces cerevisiae (baker's yeast) / Strain: NZ-16|
|Source (engineered)||Expression System: Saccharomyces cerevisiae (baker's yeast)|
|Source (natural)||Location in cell: nucleus|
-Component #2: nucleic-acid, DNA-RNA elongation scaffold
|nucleic acid||Name: DNA-RNA elongation scaffold / a.k.a: DNA-RNA elongation scaffold / Class: DNA/RNA / Structure: OTHER / Synthetic: Yes|
|Source||Species: synthetic construct (others)|
|Specimen||Specimen state: Particle / Method: cryo EM|
|Sample solution||Specimen conc.: 0.1 mg/mL|
Buffer solution: 20mM Hepes pH 7.8, 50 mM Ammonium Sulfate, 0.1 mM MgCl2, 0.01 mM ZnCl2, 5mM DTT
|Support film||Quantifoil R3/3 2nm carbon pre-coated holey grids|
|Vitrification||Instrument: FEI VITROBOT / Cryogen name: ETHANE / Temperature: 184 K / Humidity: 95 % / Details: Vitrification instrument: Vitrobot, FEI|
-Electron microscopy imaging
Model: Tecnai F20 / Image courtesy: FEI Company
|Imaging||Microscope: FEI TECNAI F20|
|Electron gun||Electron source: LAB6 / Accelerating voltage: 120 kV / Illumination mode: OTHER|
|Lens||Imaging mode: OTHER|
|Specimen Holder||Holder: Eucentric / Model: GATAN LIQUID NITROGEN|
|Processing||Method: single particle reconstruction / Number of projections: 11965 / Applied symmetry: C1 (asymmetric)|
|3D reconstruction||Resolution: 19 Å / Resolution method: FSC 0.5|
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