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- EMDB-4149: Structure of RNA polymerase I transcribing rDNA genes -

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Basic information

Entry
Database: EMDB / ID: EMD-4149
TitleStructure of RNA polymerase I transcribing rDNA genes
Map data
SampleRNA polymerase I
Biological speciesSaccharomyces cerevisiae (baker's yeast)
Methodsubtomogram averaging / cryo EM / Resolution: 29 Å
AuthorsNeyer S / Kunz M / Geiss C / Hantsche M / Hodirnau VV / Seybert A / Engel C / Scheffer MP / Cramer P / Frangakis AS
CitationJournal: 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.
History
DepositionOct 15, 2016-
Header (metadata) releaseNov 16, 2016-
Map releaseNov 23, 2016-
UpdateNov 23, 2016-
Current statusNov 23, 2016Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.27
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.27
  • Imaged by UCSF Chimera
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Structure viewerEM map:
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Supplemental images

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Map

FileDownload / File: emd_4149.map.gz / Format: CCP4 / Size: 1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
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AxesZ (Sec.)Y (Row.)X (Col.)
4 Å/pix.
x 64 pix.
= 256. Å
4 Å/pix.
x 64 pix.
= 256. Å
4 Å/pix.
x 64 pix.
= 256. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 4 Å
Density
Contour LevelBy AUTHOR: 0.27 / Movie #1: 0.27
Minimum - Maximum-6.8316064 - 7.7238283
Average (Standard dev.)0.00000041121862 (±0.9876134)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions646464
Spacing646464
CellA=B=C: 256.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z444
M x/y/z646464
origin x/y/z0.0000.0000.000
length x/y/z256.000256.000256.000
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ129141209
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS646464
D min/max/mean-6.8327.7240.000

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Supplemental data

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Sample components

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Entire RNA polymerase I

EntireName: RNA polymerase I / Number of components: 1

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Component #1: protein, RNA polymerase I

ProteinName: RNA polymerase I / Recombinant expression: No
SourceSpecies: Saccharomyces cerevisiae (baker's yeast)

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Experimental details

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Sample preparation

SpecimenSpecimen state: Cell / Method: cryo EM
Sample solutionpH: 9
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
ImagingMicroscope: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 1.2 e/Å2 / Illumination mode: FLOOD BEAM
LensImaging mode: BRIGHT FIELD
Specimen HolderModel: OTHER
CameraDetector: GATAN K2 SUMMIT (4k x 4k)

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Image processing

ProcessingMethod: subtomogram averaging / Number of subtomograms: 225
3D reconstructionResolution: 29 Å / Resolution method: FSC 0.5 CUT-OFF

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