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- EMDB-15612: RNA polymerase at U-rich pause bound to regulatory RNA putL - Pau... -

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Basic information

Entry
Database: EMDB / ID: EMD-15612
TitleRNA polymerase at U-rich pause bound to regulatory RNA putL - Pause-prone, closed clamp state
Map dataRNA polymerase at U-rich pause bound to regulatory RNA putL - Pause-prone, closed clamp state. Sharp map
Sample
  • Complex: Escherichia coli RNA polymerase putL complex - active closed clamp state
KeywordsRNA polymerase / transcriptional pausing / transcription termination / regulatory RNA / TRANSCRIPTION
Biological speciesEscherichia coli K-12 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.28 Å
AuthorsWeixlbaumer A / Dey S
Funding supportEuropean Union, France, 4 items
OrganizationGrant numberCountry
European Research Council (ERC)679734European Union
French Infrastructure for Integrated Structural Biology (FRISBI)ANR-10-INBS-05 France
Agence Nationale de la Recherche (ANR)ANR-10-LABX-0030-INRT France
Agence Nationale de la Recherche (ANR)ANR-10-IDEX-0002-02 France
CitationJournal: Mol Cell / Year: 2022
Title: Structural insights into RNA-mediated transcription regulation in bacteria.
Authors: Sanjay Dey / Claire Batisse / Jinal Shukla / Michael W Webster / Maria Takacs / Charlotte Saint-André / Albert Weixlbaumer /
Abstract: RNA can regulate its own synthesis without auxiliary proteins. For example, U-rich RNA sequences signal RNA polymerase (RNAP) to pause transcription and are required for transcript release at ...RNA can regulate its own synthesis without auxiliary proteins. For example, U-rich RNA sequences signal RNA polymerase (RNAP) to pause transcription and are required for transcript release at intrinsic terminators in all kingdoms of life. In contrast, the regulatory RNA putL suppresses pausing and termination in cis. However, how nascent RNA modulates its own synthesis remains largely unknown. We present cryo-EM reconstructions of RNAP captured during transcription of putL variants or an unrelated sequence at a U-rich pause site. Our results suggest how putL suppresses pausing and promotes its synthesis. We demonstrate that transcribing a U-rich sequence, a ubiquitous trigger of intrinsic termination, promotes widening of the RNAP nucleic-acid-binding channel. Widening destabilizes RNAP interactions with DNA and RNA to facilitate transcript dissociation reminiscent of intrinsic transcription termination. Surprisingly, RNAP remains bound to DNA after transcript release. Our results provide the structural framework to understand RNA-mediated intrinsic transcription termination.
History
DepositionAug 19, 2022-
Header (metadata) releaseOct 19, 2022-
Map releaseOct 19, 2022-
UpdateDec 13, 2023-
Current statusDec 13, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_15612.png

Downloads & links

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Map

FileDownload / File: emd_15612.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationRNA polymerase at U-rich pause bound to regulatory RNA putL - Pause-prone, closed clamp state. Sharp map
Voxel sizeX=Y=Z: 0.862 Å
Density
Contour LevelBy AUTHOR: 0.095
Minimum - Maximum-0.14690803 - 0.38197315
Average (Standard dev.)0.00108842 (±0.015863234)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions360360360
Spacing360360360
CellA=B=C: 310.32 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: RNA polymerase at U-rich pause bound to regulatory...

Fileemd_15612_additional_1.map
AnnotationRNA polymerase at U-rich pause bound to regulatory RNA putL - Pause-prone, closed clamp state
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_15612_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_15612_half_map_2.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 : Escherichia coli RNA polymerase putL complex - active closed clam...

EntireName: Escherichia coli RNA polymerase putL complex - active closed clamp state
Components
  • Complex: Escherichia coli RNA polymerase putL complex - active closed clamp state

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Supramolecule #1: Escherichia coli RNA polymerase putL complex - active closed clam...

SupramoleculeName: Escherichia coli RNA polymerase putL complex - active closed clamp state
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#7
Source (natural)Organism: Escherichia coli K-12 (bacteria)
Molecular weightTheoretical: 0.435 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

Concentration12 mg/mL
BufferpH: 8
Details: 20 mM Tris-glutamate pH 8.0, 50 mM K-glutamate, 10 mM Mg-glutamate, 0.001 mM ZnCl2, 2mM DTT
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Support film - Material: GOLD / Support film - topology: HOLEY
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 283 K / Instrument: FEI VITROBOT MARK IV
Details: Quantifoil UltrAuFoil R1.2/1.3 300 mesh holey gold grids were plasma cleaned on a Model 1070 (Fischione Instruments) for 30 sec at 70% power and with an 80% Argon and 20% Oxygen mixture ...Details: Quantifoil UltrAuFoil R1.2/1.3 300 mesh holey gold grids were plasma cleaned on a Model 1070 (Fischione Instruments) for 30 sec at 70% power and with an 80% Argon and 20% Oxygen mixture prior to the application of 0.004 ml of sample. Grids were plunge frozen into liquid ethane using a Vitrobot mark IV (FEI) with 95% chamber humidity at 283K..
DetailsThe co-transcriptionally halted complexes for cryo-EM analysis were prepared in vitro by mixing E. coli RNAP holoenzyme with templates containing the DNA base analogue isoG. The RNAP ECs halted at the U-rich pause (G93). The complexes were prepared in 0.03 ml reactions and contained: 0.02 mM of E. coli RNAP, 0.080 mM of E. coli Sigma70, 0.02 mM of template DNA, 20 mM Tris-glutamate pH 8.0, 50 mM K-glutamate, 10 mM Mg-glutamate, 0.001 mM ZnCl2, 2mM DTT and 6 mM of each ATP, GTP, CTP and UTP. Initially, all the components except NTPs were added and incubated for 10 min at 310K. To allow transcription elongation, NTPs were added and the sample was incubated for 20 to 40 min at 310K. The final products were purified on a Superose 6 Increase 3.2/300 gel filtration column equilibrated in Glutamate buffer. Samples were concentrated to 10-12 mg/mL using an Amicon Ultra 0.5mL centrifugal filter unit (30 KDa MWCO). Before grid freezing, 8 mM of CHAPSO was added to freshly prepared sample to overcome preferred particle orientation.

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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: 2.5 µm / Nominal defocus min: 0.8 µm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 1 / Average exposure time: 2.2 sec. / Average electron dose: 52.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionResolution.type: BY AUTHOR / Resolution: 4.28 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.3) / Number images used: 37763
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

6alh


Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Target criteria: map cross correlation

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