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- EMDB-5176: Frozen hydrated map of the yeast TFIID-TFIIA-Rap1-DNA complex -

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Basic information

Entry
Database: EMDB / ID: EMD-5176
TitleFrozen hydrated map of the yeast TFIID-TFIIA-Rap1-DNA complex
Map dataYeast TFIID in complex with Rap1 and DNATranscription factor II D
Sample
  • Sample: HA-tag purified yeast TFIID
  • Protein or peptide: Rap1
  • Protein or peptide: TFIIDTranscription factor II D
KeywordsTranscription / general transcription factor / TFIID / transcription initiation / transciption activator / TFIIA / Rap1
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 24.5 Å
AuthorsPapai G / Tripathi MK / Ruhlmann C / Layer JH / Weil PA / Schultz P
CitationJournal: Nature / Year: 2010
Title: TFIIA and the transactivator Rap1 cooperate to commit TFIID for transcription initiation.
Authors: Gabor Papai / Manish K Tripathi / Christine Ruhlmann / Justin H Layer / P Anthony Weil / Patrick Schultz /
Abstract: Transcription of eukaryotic messenger RNA (mRNA) encoding genes by RNA polymerase II (Pol II) is triggered by the binding of transactivating proteins to enhancer DNA, which stimulates the recruitment ...Transcription of eukaryotic messenger RNA (mRNA) encoding genes by RNA polymerase II (Pol II) is triggered by the binding of transactivating proteins to enhancer DNA, which stimulates the recruitment of general transcription factors (TFIIA, B, D, E, F, H) and Pol II on the cis-linked promoter, leading to pre-initiation complex formation and transcription. In TFIID-dependent activation pathways, this general transcription factor containing TATA-box-binding protein is first recruited on the promoter through interaction with activators and cooperates with TFIIA to form a committed pre-initiation complex. However, neither the mechanisms by which activation signals are communicated between these factors nor the structural organization of the activated pre-initiation complex are known. Here we used cryo-electron microscopy to determine the architecture of nucleoprotein complexes composed of TFIID, TFIIA, the transcriptional activator Rap1 and yeast enhancer-promoter DNA. These structures revealed the mode of binding of Rap1 and TFIIA to TFIID, as well as a reorganization of TFIIA induced by its interaction with Rap1. We propose that this change in position increases the exposure of TATA-box-binding protein within TFIID, consequently enhancing its ability to interact with the promoter. A large Rap1-dependent DNA loop forms between the activator-binding site and the proximal promoter region. This loop is topologically locked by a TFIIA-Rap1 protein bridge that folds over the DNA. These results highlight the role of TFIIA in transcriptional activation, define a molecular mechanism for enhancer-promoter communication and provide structural insights into the pathways of intramolecular communication that convey transcription activation signals through the TFIID complex.
History
DepositionMar 26, 2010-
Header (metadata) releaseApr 14, 2010-
Map releaseJun 21, 2010-
UpdateJun 21, 2010-
Current statusJun 21, 2010Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 2
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 2
  • Imaged by UCSF Chimera
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Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

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Map

FileDownload / File: emd_5176.map.gz / Format: CCP4 / Size: 15.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationYeast TFIID in complex with Rap1 and DNA
Voxel sizeX=Y=Z: 2.6 Å
Density
Contour LevelBy AUTHOR: 2.0 / Movie #1: 2
Minimum - Maximum-16.079799999999999 - 54.1081
Average (Standard dev.)-0.000000000139934 (±2.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-80-79-80
Dimensions160160160
Spacing160160160
CellA=B=C: 416 Å
α=β=γ: 90 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.62.62.6
M x/y/z160160160
origin x/y/z0.0000.0000.000
length x/y/z416.000416.000416.000
α/β/γ90.00090.00090.000
start NX/NY/NZ-34-26-72
NX/NY/NZ6953145
MAP C/R/S123
start NC/NR/NS-79-80-80
NC/NR/NS160160160
D min/max/mean-16.08054.108-0.000

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Supplemental data

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Sample components

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Entire : HA-tag purified yeast TFIID

EntireName: HA-tag purified yeast TFIID
Components
  • Sample: HA-tag purified yeast TFIID
  • Protein or peptide: Rap1
  • Protein or peptide: TFIIDTranscription factor II D

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Supramolecule #1000: HA-tag purified yeast TFIID

SupramoleculeName: HA-tag purified yeast TFIID / type: sample / ID: 1000 / Number unique components: 3
Molecular weightExperimental: 1 MDa

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Macromolecule #1: Rap1

MacromoleculeName: Rap1 / type: protein_or_peptide / ID: 1 / Name.synonym: Rap1 / Number of copies: 1 / Oligomeric state: Monomer / Recombinant expression: Yes / Database: NCBI
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: Yeast / Organelle: Nucleus / Location in cell: Nucleus

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Macromolecule #2: TFIID

MacromoleculeName: TFIID / type: protein_or_peptide / ID: 2 / Name.synonym: TFIID / Number of copies: 1 / Oligomeric state: Monomer / Recombinant expression: Yes / Database: NCBI
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: Yeast / Organelle: Nucleus / Location in cell: Nucleus

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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

Concentration0.165 mg/mL
BufferpH: 7.9 / Details: 10 mM Tris-HCl pH 7.9, 300 mM KOAc and 5 mM MgCl2
GridDetails: 300 mesh Cu/Rh holey grid
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 80 K / Instrument: OTHER / Details: Vitrification instrument: Vitrobot

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Electron microscopy

MicroscopeFEI POLARA 300
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 38950 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.0 mm / Nominal magnification: 39000
Sample stageSpecimen holder: Eucentric / Specimen holder model: OTHER
TemperatureAverage: 81 K
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: PRIMESCAN / Digitization - Sampling interval: 5.1 µm / Number real images: 90 / Average electron dose: 16 e/Å2 / Od range: 1.4
Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: Each particle
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 24.5 Å / Resolution method: OTHER / Software - Name: Imagic, Spider
Details: The map was reconstructed from 3-D MSA separated dataset.
Number images used: 26901

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