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- EMDB-14450: Pol III PTC - Full transcription bubble -

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Basic information

Entry
Database: EMDB / ID: EMD-14450
TitlePol III PTC - Full transcription bubble
Map dataPol III PTC NTPs DNA/RNA classified Sharpened with LocalDeblur
Sample
  • Complex: Yeast RNA Polymerase III PTC + NTPs, Full transcription bubble
KeywordsRNA synthesis / short RNAs / termination / TRANSCRIPTION
Biological speciesSaccharomyces cerevisiae W303 (yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsGirbig M / Mueller CW
Funding support Germany, 1 items
OrganizationGrant numberCountry
Boehringer Ingelheim Fonds (BIF) Germany
CitationJournal: Cell Rep / Year: 2022
Title: Architecture of the yeast Pol III pre-termination complex and pausing mechanism on poly(dT) termination signals.
Authors: Mathias Girbig / Juanjuan Xie / Helga Grötsch / Domenico Libri / Odil Porrua / Christoph W Müller /
Abstract: RNA polymerase (Pol) III is specialized to transcribe short, abundant RNAs, for which it terminates transcription on polythymine (dT) stretches on the non-template (NT) strand. When Pol III reaches ...RNA polymerase (Pol) III is specialized to transcribe short, abundant RNAs, for which it terminates transcription on polythymine (dT) stretches on the non-template (NT) strand. When Pol III reaches the termination signal, it pauses and forms the pre-termination complex (PTC). Here, we report cryoelectron microscopy (cryo-EM) structures of the yeast Pol III PTC and complementary functional states at resolutions of 2.7-3.9 Å. Pol III recognizes the poly(dT) termination signal with subunit C128 that forms a hydrogen-bond network with the NT strand and, thereby, induces pausing. Mutating key interacting residues interferes with transcription termination in vitro, impairs yeast growth, and causes global termination defects in vivo, confirming our structural results. Additional cryo-EM analysis reveals that C53-C37, a Pol III subcomplex and key termination factor, participates indirectly in Pol III termination. We propose a mechanistic model of Pol III transcription termination and rationalize why Pol III, unlike Pol I and Pol II, terminates on poly(dT) signals.
History
DepositionFeb 24, 2022-
Header (metadata) releaseAug 31, 2022-
Map releaseAug 31, 2022-
UpdateDec 13, 2023-
Current statusDec 13, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_14450.png

Downloads & links

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Map

FileDownload / File: emd_14450.map.gz / Format: CCP4 / Size: 163.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationPol III PTC NTPs DNA/RNA classified Sharpened with LocalDeblur
Voxel sizeX=Y=Z: 1.041 Å
Density
Contour LevelBy AUTHOR: 0.013
Minimum - Maximum-0.078084104 - 0.68593025
Average (Standard dev.)0.0005804553 (±0.009786681)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions350350350
Spacing350350350
CellA=B=C: 364.35 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: Pol III PTC NTPs DNA/RNA classified Unsharpened map

Fileemd_14450_additional_1.map
AnnotationPol III PTC NTPs DNA/RNA classified Unsharpened map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Yeast RNA Polymerase III PTC + NTPs, Full transcription bubble

EntireName: Yeast RNA Polymerase III PTC + NTPs, Full transcription bubble
Components
  • Complex: Yeast RNA Polymerase III PTC + NTPs, Full transcription bubble

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Supramolecule #1: Yeast RNA Polymerase III PTC + NTPs, Full transcription bubble

SupramoleculeName: Yeast RNA Polymerase III PTC + NTPs, Full transcription bubble
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#20
Source (natural)Organism: Saccharomyces cerevisiae W303 (yeast)
Molecular weightTheoretical: 730 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

Concentration2.4 mg/mL
BufferpH: 7.5
Component:
ConcentrationName
0.015 MHEPES
0.15 MAmmonium sulfate
0.005 MMagnesium chloride
0.01 MDTT
0.001 MCTP, ATP, UTP
0.004 MCHAPSO
GridModel: Quantifoil R2/1 / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K2 IS (4k x 4k) / Average electron dose: 44.8 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.25 µm / Nominal defocus min: 0.75 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 538161
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

6tut

Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 23641
Initial angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION (ver. 3.1)
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION (ver. 3.1)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

6tut


Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementProtocol: OTHER

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