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- EMDB-28024: Dimerized Mediator PIC with early GTFs on a divergent promoter -

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Basic information

Entry
Database: EMDB / ID: EMD-28024
TitleDimerized Mediator PIC with early GTFs on a divergent promoter
Map dataDimerized Mediator PIC with early GTFs on a divergent promoter
Sample
  • Complex: Complex of Mediator, RNA Polymerase II, and general transcription factors (TFIIA, TFIIB, TBP, TFIIF, TFIIS) on a divergent promoter.
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsubtomogram averaging / cryo EM / Resolution: 36.0 Å
AuthorsPalao III L
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS) United States
CitationJournal: Mol Cell / Year: 2023
Title: Structural basis of a transcription pre-initiation complex on a divergent promoter.
Authors: Jose J Gorbea Colón / Leon Palao / Shin-Fu Chen / Hee Jong Kim / Laura Snyder / Yi-Wei Chang / Kuang-Lei Tsai / Kenji Murakami /
Abstract: Most eukaryotic promoter regions are divergently transcribed. As the RNA polymerase II pre-initiation complex (PIC) is intrinsically asymmetric and responsible for transcription in a single ...Most eukaryotic promoter regions are divergently transcribed. As the RNA polymerase II pre-initiation complex (PIC) is intrinsically asymmetric and responsible for transcription in a single direction, it is unknown how divergent transcription arises. Here, the Saccharomyces cerevisiae Mediator complexed with a PIC (Med-PIC) was assembled on a divergent promoter and analyzed by cryoelectron microscopy. The structure reveals two distinct Med-PICs forming a dimer through the Mediator tail module, induced by a homodimeric activator protein localized near the dimerization interface. The tail dimer is associated with ∼80-bp upstream DNA, such that two flanking core promoter regions are positioned and oriented in a suitable form for PIC assembly in opposite directions. Also, cryoelectron tomography visualized the progress of the PIC assembly on the two core promoter regions, providing direct evidence for the role of the Med-PIC dimer in divergent transcription.
History
DepositionSep 2, 2022-
Header (metadata) releaseFeb 15, 2023-
Map releaseFeb 15, 2023-
UpdateMar 8, 2023-
Current statusMar 8, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_28024.png

Downloads & links

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Map

FileDownload / File: emd_28024.map.gz / Format: CCP4 / Size: 22.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationDimerized Mediator PIC with early GTFs on a divergent promoter
Voxel sizeX=Y=Z: 3.352 Å
Density
Contour LevelBy AUTHOR: 140.0
Minimum - Maximum0.00048828125 - 255.00024
Average (Standard dev.)104.49863 (±12.406478)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions180180180
Spacing180180180
CellA=B=C: 603.36 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Half Map 1

Fileemd_28024_half_map_1.map
AnnotationHalf Map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half Map 2

Fileemd_28024_half_map_2.map
AnnotationHalf Map 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Complex of Mediator, RNA Polymerase II, and general transcription...

EntireName: Complex of Mediator, RNA Polymerase II, and general transcription factors (TFIIA, TFIIB, TBP, TFIIF, TFIIS) on a divergent promoter.
Components
  • Complex: Complex of Mediator, RNA Polymerase II, and general transcription factors (TFIIA, TFIIB, TBP, TFIIF, TFIIS) on a divergent promoter.

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Supramolecule #1: Complex of Mediator, RNA Polymerase II, and general transcription...

SupramoleculeName: Complex of Mediator, RNA Polymerase II, and general transcription factors (TFIIA, TFIIB, TBP, TFIIF, TFIIS) on a divergent promoter.
type: complex / ID: 1 / Chimera: Yes / Parent: 0
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)

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Experimental details

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Structure determination

Methodcryo EM
Processingsubtomogram averaging
Aggregation stateparticle

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Sample preparation

Concentration0.1 mg/mL
BufferpH: 7.6
Component:
ConcentrationFormulaName
20.0 mMC8H18N2O4SHEPES
80.0 mMCH3COOKPotassium Acetate
2.0 mM(CH3COO)2MgMagnesium Acetate

Details: 20mM HEPES pH7.6, 2mM Magnesium Acetate, 80mM Potassium Acetate, 10nm gold beads
GridModel: Quantifoil R2/2 / Material: COPPER / Mesh: 200 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec. / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE-PROPANE / Chamber humidity: 65 % / Chamber temperature: 298 K / Instrument: LEICA EM CPC
DetailsSamples were chemically stabilized by fixation in glycerol gradients with increasing concentrations of glutaraldehyde (0-0.125%). Peak gradient fractions containing glutaraldhyde were quenched upon the addition of 50 mM glycine (pH 7.6) and 10nm gold beads were added before flash-freezing and storage under liquid nitrogen.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Illumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 5.0 µm / Nominal defocus min: 0.3 µm / Nominal magnification: 53000
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 1.95 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

ExtractionNumber tomograms: 55 / Number images used: 819 / Software - Name: IMOD
Final angle assignmentType: PROJECTION MATCHING
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 36.0 Å / Resolution method: FSC 0.143 CUT-OFF / Number subtomograms used: 819
FSC plot (resolution estimation)

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