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- EMDB-15613: RNA polymerase bound to purified in vitro transcribed regulatory ... -

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Entry
Database: EMDB / ID: EMD-15613
TitleRNA polymerase bound to purified in vitro transcribed regulatory RNA putL - inactive, open clamp state
Map data
Sample
  • Complex: Escherichia coli RNA polymerase and in vitro transcribed and purified putL RNA complex - inactive, open clamp state
KeywordsRNA polymerase / transcriptional pausing / transcription termination / regulatory RNA / TRANSCRIPTION
Biological speciesEscherichia coli K-12 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.4 Å
AuthorsDey S / Weixlbaumer A
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_15613.png

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Map

FileDownload / File: emd_15613.map.gz / Format: CCP4 / Size: 83.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.1 Å
Density
Contour LevelBy AUTHOR: 0.435
Minimum - Maximum-0.68889713 - 1.4701662
Average (Standard dev.)0.007449207 (±0.06649663)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions280280280
Spacing280280280
CellA=B=C: 308.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: #1

Fileemd_15613_additional_1.map
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AxesZYX

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Half map: #1

Fileemd_15613_half_map_1.map
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Histogram (log scale)

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Half map: #2

Fileemd_15613_half_map_2.map
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Sample components

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Entire : Escherichia coli RNA polymerase and in vitro transcribed and puri...

EntireName: Escherichia coli RNA polymerase and in vitro transcribed and purified putL RNA complex - inactive, open clamp state
Components
  • Complex: Escherichia coli RNA polymerase and in vitro transcribed and purified putL RNA complex - inactive, open clamp state

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Supramolecule #1: Escherichia coli RNA polymerase and in vitro transcribed and puri...

SupramoleculeName: Escherichia coli RNA polymerase and in vitro transcribed and purified putL RNA complex - inactive, open clamp state
type: complex / ID: 1 / Parent: 0
Source (natural)Organism: Escherichia coli K-12 (bacteria)
Molecular weightTheoretical: 435 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

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: C-flat-1.2/1.3 / Material: GOLD / Mesh: 300
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 complex for cryo-EM analysis was prepared in vitro by mixing E. coli RNAP with DNA oligonucleotides and in vitro transcribed and purified putL RNA to mimic an RNAP EC halted at the U-rich pause (G93). The complex was prepared in 20 mM Tris-glutamate pH 8.0, 50 mM K-glutamate, 10 mM Mg-glutamate, 0.001 mM ZnCl2, and 2mM DTT. The final complex was 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
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
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

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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: 4.4 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 17816
FSC plot (resolution estimation)

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