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- EMDB-17877: Structure of Rho pentamer in complex with Rof -

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Basic information

Entry
Database: EMDB / ID: EMD-17877
TitleStructure of Rho pentamer in complex with Rof
Map dataEM map
Sample
  • Complex: Rho-Rof complex
    • Protein or peptide: Transcription termination factor Rho
    • Protein or peptide: Protein rof
KeywordsTranscription termination factor Rho Inhibition of termination Sm-like protein Rof Rho regulation / TRANSCRIPTION
Function / homology
Function and homology information


regulation of termination of DNA-templated transcription / ATP-dependent activity, acting on RNA / transcription antitermination / DNA-templated transcription termination / helicase activity / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / ATP hydrolysis activity / RNA binding / ATP binding / identical protein binding ...regulation of termination of DNA-templated transcription / ATP-dependent activity, acting on RNA / transcription antitermination / DNA-templated transcription termination / helicase activity / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / ATP hydrolysis activity / RNA binding / ATP binding / identical protein binding / membrane / cytosol
Similarity search - Function
Modulator of Rho-dependent transcription termination / Rof-like superfamily / Modulator of Rho-dependent transcription termination (ROF) / Rof/RNase P-like / Transcription termination factor Rho / Rho termination factor, N-terminal / Rho termination factor, RNA-binding domain / Transcription termination factor Rho, ATP binding domain / Rho termination factor, RNA-binding domain / Rho termination factor, N-terminal domain ...Modulator of Rho-dependent transcription termination / Rof-like superfamily / Modulator of Rho-dependent transcription termination (ROF) / Rof/RNase P-like / Transcription termination factor Rho / Rho termination factor, N-terminal / Rho termination factor, RNA-binding domain / Transcription termination factor Rho, ATP binding domain / Rho termination factor, RNA-binding domain / Rho termination factor, N-terminal domain / Rho RNA-binding domain profile. / Rho termination factor, N-terminal domain / Rho termination factor, N-terminal domain superfamily / Cold shock domain / Cold shock protein domain / ATPase, F1/V1/A1 complex, alpha/beta subunit, nucleotide-binding domain / ATP synthase alpha/beta family, nucleotide-binding domain / Nucleic acid-binding, OB-fold / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Protein rof / Transcription termination factor Rho
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.0 Å
AuthorsSaid N / Hilal T / Wahl MC
Funding support Germany, 2 items
OrganizationGrant numberCountry
German Research Foundation (DFG)392923329 Germany
German Research Foundation (DFG)433623608 Germany
CitationJournal: Nat Commun / Year: 2024
Title: Sm-like protein Rof inhibits transcription termination factor ρ by binding site obstruction and conformational insulation.
Authors: Nelly Said / Mark Finazzo / Tarek Hilal / Bing Wang / Tim Luca Selinger / Daniela Gjorgjevikj / Irina Artsimovitch / Markus C Wahl /
Abstract: Transcription termination factor ρ is a hexameric, RNA-dependent NTPase that can adopt active closed-ring and inactive open-ring conformations. The Sm-like protein Rof, a homolog of the RNA ...Transcription termination factor ρ is a hexameric, RNA-dependent NTPase that can adopt active closed-ring and inactive open-ring conformations. The Sm-like protein Rof, a homolog of the RNA chaperone Hfq, inhibits ρ-dependent termination in vivo but recapitulation of this activity in vitro has proven difficult and the precise mode of Rof action is presently unknown. Here, our cryo-EM structures of ρ-Rof and ρ-RNA complexes show that Rof undergoes pronounced conformational changes to bind ρ at the protomer interfaces, undercutting ρ conformational dynamics associated with ring closure and occluding extended primary RNA-binding sites that are also part of interfaces between ρ and RNA polymerase. Consistently, Rof impedes ρ ring closure, ρ-RNA interactions and ρ association with transcription elongation complexes. Structure-guided mutagenesis coupled with functional assays confirms that the observed ρ-Rof interface is required for Rof-mediated inhibition of cell growth and ρ-termination in vitro. Bioinformatic analyses reveal that Rof is restricted to Pseudomonadota and that the ρ-Rof interface is conserved. Genomic contexts of rof differ between Enterobacteriaceae and Vibrionaceae, suggesting distinct modes of Rof regulation. We hypothesize that Rof and other cellular anti-terminators silence ρ under diverse, but yet to be identified, stress conditions when unrestrained transcription termination by ρ may be detrimental.
History
DepositionJul 14, 2023-
Header (metadata) releaseApr 17, 2024-
Map releaseApr 17, 2024-
UpdateApr 24, 2024-
Current statusApr 24, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_17877.png

Downloads & links

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Map

FileDownload / File: emd_17877.map.gz / Format: CCP4 / Size: 216 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationEM map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 384 pix.
= 319.488 Å		0.83 Å/pix.
x 384 pix.
= 319.488 Å		0.83 Å/pix.
x 384 pix.
= 319.488 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.832 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0982
Minimum - Maximum-0.0 - 1.8491497
Average (Standard dev.)0.004409621 (±0.037723973)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions384384384
Spacing384384384
CellA=B=C: 319.488 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: EM half map B

Fileemd_17877_half_map_1.map
AnnotationEM half map B
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: EM half map A

Fileemd_17877_half_map_2.map
AnnotationEM half map A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Rho-Rof complex

EntireName: Rho-Rof complex
Components
  • Complex: Rho-Rof complex
    • Protein or peptide: Transcription termination factor Rho
    • Protein or peptide: Protein rof

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Supramolecule #1: Rho-Rof complex

SupramoleculeName: Rho-Rof complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all / Details: Rho pentamer in complex with five Rof proteins
Source (natural)Organism: Escherichia coli (E. coli)

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Macromolecule #1: Transcription termination factor Rho

MacromoleculeName: Transcription termination factor Rho / type: protein_or_peptide / ID: 1 / Number of copies: 5 / Enantiomer: LEVO
EC number: Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 47.070168 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MNLTELKNTP VSELITLGEN MGLENLARMR KQDIIFAILK QHAKSGEDIF GDGVLEILQD GFGFLRSADS SYLAGPDDIY VSPSQIRRF NLRTGDTISG KIRPPKEGER YFALLKVNEV NFDKPENARN KILFENLTPL HANSRLRMER GNGSTEDLTA R VLDLASPI ...String:
MNLTELKNTP VSELITLGEN MGLENLARMR KQDIIFAILK QHAKSGEDIF GDGVLEILQD GFGFLRSADS SYLAGPDDIY VSPSQIRRF NLRTGDTISG KIRPPKEGER YFALLKVNEV NFDKPENARN KILFENLTPL HANSRLRMER GNGSTEDLTA R VLDLASPI GRGQRGLIVA PPKAGKTMLL QNIAQSIAYN HPDCVLMVLL IDERPEEVTE MQRLVKGEVV ASTFDEPASR HV QVAEMVI EKAKRLVEHK KDVIILLDSI TRLARAYNTV VPASGKVLTG GVDANALHRP KRFFGAARNV EEGGSLTIIA TAL IDTGSK MDEVIYEEFK GTGNMELHLS RKIAEKRVFP AIDYNRSGTR KEELLTTQEE LQKMWILRKI IHPMGEIDAM EFLI NKLAM TKTNDDFFEM MKRS

UniProtKB: Transcription termination factor Rho

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Macromolecule #2: Protein rof

MacromoleculeName: Protein rof / type: protein_or_peptide / ID: 2 / Number of copies: 4 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 9.618758 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
AMGNDTYQPI NCDDYDNLEL ACQHHLMLTL ELKDGEKLQA KASDLVSRKN VEYLVVEAAG ETRELRLDKI TSFSHPEIGT VVVSES

UniProtKB: Protein rof

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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

BufferpH: 8
VitrificationCryogen name: NITROGEN / Chamber humidity: 100 % / Chamber temperature: 283 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
SoftwareName: EPU (ver. 2.8)
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: COUNTING / Number grids imaged: 1 / Number real images: 1555 / Average electron dose: 40.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.9 µm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 619090
Startup modelType of model: OTHER
Final reconstructionAlgorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 3.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.3.1) / Number images used: 285647
Initial angle assignmentType: RANDOM ASSIGNMENT / Software - Name: cryoSPARC (ver. 3.3.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.3.1)
Final 3D classificationSoftware - Name: cryoSPARC (ver. 3.3.1)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelChain - Chain ID: A / Chain - Residue range: 1-419 / Chain - Source name: Other / Chain - Initial model type: experimental model / Details: own experimental data
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Overall B value: 106
Output model

PDB-8ptp:
Structure of Rho pentamer in complex with Rof

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