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- EMDB-2923: Electron cryo-microscopy of a transcribing Pol IIp complex -

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Basic information

Entry
Database: EMDB / ID: 2923
TitleElectron cryo-microscopy of a transcribing Pol IIp complex
Map dataReconstruction of a transcribing Pol IIp complex
Sampletranscribing Pol IIp complex from yeast
  • DNA-directed RNA polymerase IIPolymerase
  • nucleic-acidNucleic acid
KeywordsTranscription / pre-mRNA capping
SourceSaccharomyces cerevisiae (baker's yeast)
Methodsingle particle reconstruction / cryo EM / 7.2 Å resolution
AuthorsMartinez-Rucobo FW / Kohler R / van de Waterbeemd M / Heck AJR / Hemann M / Herzog F / Stark H / Cramer P
CitationJournal: Mol. Cell / Year: 2015
Title: Molecular Basis of Transcription-Coupled Pre-mRNA Capping.
Authors: Fuensanta W Martinez-Rucobo / Rebecca Kohler / Michiel van de Waterbeemd / Albert J R Heck / Matthias Hemann / Franz Herzog / Holger Stark / Patrick Cramer
Abstract: Capping is the first step in pre-mRNA processing, and the resulting 5'-RNA cap is required for mRNA splicing, export, translation, and stability. Capping is functionally coupled to transcription by ...Capping is the first step in pre-mRNA processing, and the resulting 5'-RNA cap is required for mRNA splicing, export, translation, and stability. Capping is functionally coupled to transcription by RNA polymerase (Pol) II, but the coupling mechanism remains unclear. We show that efficient binding of the capping enzyme (CE) to transcribing, phosphorylated yeast Pol II (Pol IIp) requires nascent RNA with an unprocessed 5'-triphosphate end. The transcribing Pol IIp-CE complex catalyzes the first two steps of capping, and its analysis by mass spectrometry, cryo-electron microscopy, and protein crosslinking revealed the molecular basis for transcription-coupled pre-mRNA capping. CE docks to the Pol II wall and spans the end of the RNA exit tunnel to position the CE active sites for sequential binding of the exiting RNA 5' end. Thus, the RNA 5' end triggers its own capping when it emerges from Pol II, to ensure seamless RNA protection from 5'-exonucleases during early transcription.
DateDeposition: Feb 27, 2015 / Header (metadata) release: Apr 1, 2015 / Map release: Sep 23, 2015 / Last update: Sep 23, 2015

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.04
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.04
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

Fileemd_2923.map.gz (map file in CCP4 format, 12665 KB)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
148 pix
2 Å/pix.
= 296. Å
148 pix
2 Å/pix.
= 296. Å
148 pix
2 Å/pix.
= 296. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2 Å
Density
Contour Level:0.04 (by author), 0.04 (movie #1):
Minimum - Maximum-0.31250411 - 0.41651475
Average (Standard dev.)0.00077728 (0.01971558)
Details

EMDB XML:

Space Group Number1
Map Geometry
Axis orderXYZ
Dimensions148148148
Origin000
Limit147147147
Spacing148148148
CellA=B=C: 296 Å
α=β=γ: 90 deg.

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z222
M x/y/z148148148
origin x/y/z0.0000.0000.000
length x/y/z296.000296.000296.000
α/β/γ90.00090.00090.000
start NX/NY/NZ
NX/NY/NZ
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS148148148
D min/max/mean-0.3130.4170.001

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Supplemental data

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Sample components

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Entire transcribing Pol IIp complex from yeast

EntireName: transcribing Pol IIp complex from yeast / Number of components: 2
Oligomeric State: One monomer of Pol IIp contains Pol IIp contains 1 DNA/RNA scaffold of 2 DNA and 1 RNA molecules
MassTheoretical: 540 kDa / Experimental: 540 kDa / Measured by: native mass spectroscopy

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Component #1: protein, DNA-directed RNA polymerase II

ProteinName: DNA-directed RNA polymerase IIPolymerase / a.k.a: RNA polymerase II / Oligomeric Details: Monomer / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 520 kDa / Experimental: 520 kDa
SourceSpecies: Saccharomyces cerevisiae (baker's yeast)

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Component #2: nucleic-acid, DNA-RNA Scaffold

Nucleic-acidName: DNA-RNA Scaffold / a.k.a: template DNA, non-template DNA, mRNA / Class: DNA/RNA / Structure: OTHER / Synthetic: Yes
MassTheoretical: 20 kDa / Experimental: 20 kDa
SourceSpecies: Saccharomyces cerevisiae (baker's yeast)

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Experimental details

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Sample preparation

SpecimenSpecimen state: particle / Method: cryo EM
Sample solutionSpecimen conc.: 0.25 mg/ml / Buffer solution: 5mM HEPES, 100mM KCl, 3mM DTT / pH: 7.25
Support film200 mesh grid with thin carbon support
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
ImagingMicroscope: FEI TITAN KRIOS / Date: Jan 7, 2014
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 25 e/Å2 / Illumination mode: SPOT SCAN
LensMagnification: 74000 X (calibrated) / Cs: 0 mm / Imaging mode: BRIGHT FIELD / Defocus: 1500 - 4500 nm
Specimen HolderModel: FEI TITAN KRIOS AUTOGRID HOLDER
CameraDetector: FEI FALCON II (4k x 4k)

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C1 (asymmetric) / Number of projections: 66214
3D reconstructionSoftware: Relion / CTF correction: each particle / Resolution: 7.2 Å / Resolution method: FSC 0.143, gold-standard

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