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- EMDB-5442: Regulation of mammalian transcription by Gdown1 through a novel s... -

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Basic information

Entry
Database: EMDB / ID: EMD-5442
TitleRegulation of mammalian transcription by Gdown1 through a novel steric crosstalk revealed by cryo-EM
Map dataReconstruction of Bovine RNA polymerase II with TFIIF
Sample
  • Sample: Bovine RNA polymerase II complex
  • Protein or peptide: RNA polymerase II elongation complex
KeywordsRNA polymerase / Mediator / Gdown1 / TFIIF
Biological speciesBos taurus (cattle)
Methodsingle particle reconstruction / cryo EM / Resolution: 19.0 Å
AuthorsWu YM / Chang JW / Wang CH / Lin YC / Wu PL / Huang SH / Chang CC / Hu X / Gnatt A / Chang WH
CitationJournal: EMBO J / Year: 2012
Title: Regulation of mammalian transcription by Gdown1 through a novel steric crosstalk revealed by cryo-EM.
Authors: Yi-Min Wu / Jen-Wei Chang / Chun-Hsiung Wang / Yen-Chen Lin / Pei-lun Wu / Shih-hsin Huang / Chia-Chi Chang / Xiaopeng Hu / Averell Gnatt / Wei-hau Chang /
Abstract: In mammals, a distinct RNA polymerase II form, RNAPII(G) contains a novel subunit Gdown1 (encoded by POLR2M), which represses gene activation, only to be reversed by the multisubunit Mediator co- ...In mammals, a distinct RNA polymerase II form, RNAPII(G) contains a novel subunit Gdown1 (encoded by POLR2M), which represses gene activation, only to be reversed by the multisubunit Mediator co-activator. Here, we employed single-particle cryo-electron microscopy (cryo-EM) to disclose the architectures of RNAPII(G), RNAPII and RNAPII in complex with the transcription initiation factor TFIIF, all to ~19 Å. Difference analysis mapped Gdown1 mostly to the RNAPII Rpb5 shelf-Rpb1 jaw, supported by antibody labelling experiments. These structural features correlate with the moderate increase in the efficiency of RNA chain elongation by RNAP II(G). In addition, our updated RNAPII-TFIIF map showed that TFIIF tethers multiple regions surrounding the DNA-binding cleft, in agreement with cross-linking and biochemical mapping. Gdown1's binding sites overlap extensively with those of TFIIF, with Gdown1 sterically excluding TFIIF from RNAPII, herein demonstrated by competition assays using size exclusion chromatography. In summary, our work establishes a structural basis for Gdown1 impeding initiation at promoters, by obstruction of TFIIF, accounting for an additional dependent role of Mediator in activated transcription.
History
DepositionJun 29, 2012-
Header (metadata) releaseJul 25, 2012-
Map releaseFeb 13, 2013-
UpdateFeb 13, 2013-
Current statusFeb 13, 2013Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0024
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.0024
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

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Map

FileDownload / File: emd_5442.map.gz / Format: CCP4 / Size: 12.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of Bovine RNA polymerase II with TFIIF
Voxel sizeX=Y=Z: 1.9 Å
Density
Contour LevelBy AUTHOR: 0.0024 / Movie #1: 0.0024
Minimum - Maximum-0.00507214 - 0.00952793
Average (Standard dev.)0.00001049 (±0.00099565)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions149149149
Spacing149149149
CellA=B=C: 283.1 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.91.91.9
M x/y/z149149149
origin x/y/z0.0000.0000.000
length x/y/z283.100283.100283.100
α/β/γ90.00090.00090.000
start NX/NY/NZ-150-1500
NX/NY/NZ301301151
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS149149149
D min/max/mean-0.0050.0100.000

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Supplemental data

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Sample components

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Entire : Bovine RNA polymerase II complex

EntireName: Bovine RNA polymerase II complex
Components
  • Sample: Bovine RNA polymerase II complex
  • Protein or peptide: RNA polymerase II elongation complex

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Supramolecule #1000: Bovine RNA polymerase II complex

SupramoleculeName: Bovine RNA polymerase II complex / type: sample / ID: 1000 / Number unique components: 1
Molecular weightTheoretical: 620 KDa

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Macromolecule #1: RNA polymerase II elongation complex

MacromoleculeName: RNA polymerase II elongation complex / type: protein_or_peptide / ID: 1 / Oligomeric state: monomer / Recombinant expression: No / Database: NCBI
Source (natural)Organism: Bos taurus (cattle) / synonym: bovine / Tissue: thymus
Molecular weightTheoretical: 620 KDa

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration1 mg/mL
BufferpH: 8 / Details: 50 mM Tris-Cl, 200 mM KOAc, 5mM MgCl2, 5 mM DDT
GridDetails: 200 mesh quantifoil grid with thin carbon layer
VitrificationCryogen name: ETHANE / Chamber humidity: 80 % / Instrument: HOMEMADE PLUNGER

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Electron microscopy

MicroscopeJEOL 2100F
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: SPOT SCAN / Imaging mode: DARK FIELD
Sample stageSpecimen holder model: GATAN HELIUM
DateMar 14, 2012
Image recordingDigitization - Scanner: ZEISS SCAI / Number real images: 150 / Average electron dose: 20 e/Å2 / Bits/pixel: 16

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Image processing

CTF correctionDetails: Each particle
Final two d classificationNumber classes: 128
Final reconstructionResolution.type: BY AUTHOR / Resolution: 19.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: Spider, EMAN2 / Number images used: 24000

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