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- EMDB-29356: Focused refinement on the Brf1-TBP-DNA within TFIIIA-TFIIIC-Brf1-... -

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Basic information

Entry
Database: EMDB / ID: EMD-29356
TitleFocused refinement on the Brf1-TBP-DNA within TFIIIA-TFIIIC-Brf1-TBP complex bound to 5S rRNA gene
Map datapostprocessed map
Sample
  • Complex: TFIIIA-TFIIIC-Brf1-TBP complex bound to 5SrRNA gene
Keywordstranscription factor / TRANSCRIPTION-DNA complex
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 7.14 Å
AuthorsTalyzina A / He Y
Funding support United States, 3 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS) United States
American Cancer Society United States
National Institutes of Health/National Cancer Institute (NIH/NCI) United States
CitationJournal: Mol Cell / Year: 2023
Title: Structural basis of TFIIIC-dependent RNA polymerase III transcription initiation.
Authors: Anna Talyzina / Yan Han / Chiranjib Banerjee / Susan Fishbain / Alexis Reyes / Reza Vafabakhsh / Yuan He /
Abstract: RNA polymerase III (Pol III) is responsible for transcribing 5S ribosomal RNA (5S rRNA), tRNAs, and other short non-coding RNAs. Its recruitment to the 5S rRNA promoter requires transcription factors ...RNA polymerase III (Pol III) is responsible for transcribing 5S ribosomal RNA (5S rRNA), tRNAs, and other short non-coding RNAs. Its recruitment to the 5S rRNA promoter requires transcription factors TFIIIA, TFIIIC, and TFIIIB. Here, we use cryoelectron microscopy (cryo-EM) to visualize the S. cerevisiae complex of TFIIIA and TFIIIC bound to the promoter. Gene-specific factor TFIIIA interacts with DNA and acts as an adaptor for TFIIIC-promoter interactions. We also visualize DNA binding of TFIIIB subunits, Brf1 and TBP (TATA-box binding protein), which results in the full-length 5S rRNA gene wrapping around the complex. Our smFRET study reveals that the DNA within the complex undergoes both sharp bending and partial dissociation on a slow timescale, consistent with the model predicted from our cryo-EM results. Our findings provide new insights into the transcription initiation complex assembly on the 5S rRNA promoter and allow us to directly compare Pol III and Pol II transcription adaptations.
History
DepositionJan 3, 2023-
Header (metadata) releaseJul 19, 2023-
Map releaseJul 19, 2023-
UpdateAug 16, 2023-
Current statusAug 16, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_29356.png

Downloads & links

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Map

FileDownload / File: emd_29356.map.gz / Format: CCP4 / Size: 216 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationpostprocessed map
Voxel sizeX=Y=Z: 1.056 Å
Density
Contour LevelBy AUTHOR: 0.2
Minimum - Maximum-0.23174237 - 1.0458584
Average (Standard dev.)0.0020263614 (±0.024390297)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions384384384
Spacing384384384
CellA=B=C: 405.504 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: deepemhancer map

Fileemd_29356_additional_1.map
Annotationdeepemhancer map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half 2

Fileemd_29356_half_map_1.map
Annotationhalf 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half 1

Fileemd_29356_half_map_2.map
Annotationhalf 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : TFIIIA-TFIIIC-Brf1-TBP complex bound to 5SrRNA gene

EntireName: TFIIIA-TFIIIC-Brf1-TBP complex bound to 5SrRNA gene
Components
  • Complex: TFIIIA-TFIIIC-Brf1-TBP complex bound to 5SrRNA gene

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Supramolecule #1: TFIIIA-TFIIIC-Brf1-TBP complex bound to 5SrRNA gene

SupramoleculeName: TFIIIA-TFIIIC-Brf1-TBP complex bound to 5SrRNA gene / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#10
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 810 kDa/nm

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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

Concentration0.02 mg/mL
BufferpH: 7.9
GridModel: Quantifoil R2/1 / Support film - Material: GRAPHENE OXIDE / Support film - topology: CONTINUOUS
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 5.0 µm / Nominal defocus min: 2.0 µm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 60.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 5748589
Startup modelType of model: OTHER / Details: Map from negatively stained sample
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final 3D classificationNumber classes: 3 / Avg.num./class: 75000 / Software - Name: cryoSPARC
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final reconstructionResolution.type: BY AUTHOR / Resolution: 7.14 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 12232

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: RIGID BODY FIT

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