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- PDB-8ptn: Structure of the transcription termination factor Rho in complex ... -

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Basic information

Entry
Database: PDB / ID: 8ptn
TitleStructure of the transcription termination factor Rho in complex with Rof
Components
  • Protein rof
  • Transcription termination factor Rho
KeywordsTRANSCRIPTION / Transcription termination factor Rho Inhibition of termination Sm-like protein Rof Rho regulation
Function / homology
Function and homology information


regulation of termination of DNA-templated transcription / ATP-dependent activity, acting on RNA / transcription antitermination / helicase activity / DNA-templated transcription termination / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / ATP hydrolysis activity / RNA binding / ATP binding / membrane ...regulation of termination of DNA-templated transcription / ATP-dependent activity, acting on RNA / transcription antitermination / helicase activity / DNA-templated transcription termination / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / ATP hydrolysis activity / RNA binding / ATP binding / membrane / identical protein binding / 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 / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / Nucleic acid-binding, OB-fold / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Protein rof / Transcription termination factor Rho
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsSaid, N. / Hilal, T. / Wahl, M.C.
Funding support Germany, 2items
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, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Apr 17, 2024Provider: repository / Type: Initial release
Revision 1.1Apr 24, 2024Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Transcription termination factor Rho
B: Transcription termination factor Rho
C: Transcription termination factor Rho
D: Transcription termination factor Rho
E: Transcription termination factor Rho
F: Transcription termination factor Rho
a: Protein rof
b: Protein rof
c: Protein rof
d: Protein rof
e: Protein rof


Theoretical massNumber of molelcules
Total (without water)330,51511
Polymers330,51511
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
Transcription termination factor Rho / ATP-dependent helicase Rho


Mass: 47070.168 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: rho, nitA, psuA, rnsC, sbaA, tsu, b3783, JW3756 / Production host: Escherichia coli (E. coli)
References: UniProt: P0AG30, Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement
#2: Protein
Protein rof


Mass: 9618.758 Da / Num. of mol.: 5
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: rof, yaeO, b0189, JW0184 / Production host: Escherichia coli (E. coli) / References: UniProt: P0AFW8

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Rho-Rof complex / Type: COMPLEX / Details: Rho hexamer in complex with five Rof proteins / Entity ID: all / Source: RECOMBINANT
Molecular weightUnits: KILODALTONS/NANOMETER / Experimental value: NO
Source (natural)Organism: Escherichia coli (E. coli)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 8
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: NITROGEN / Humidity: 100 % / Chamber temperature: 283 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2000 nm / Nominal defocus min: 900 nm / Cs: 2.7 mm / Alignment procedure: ZEMLIN TABLEAU
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 40 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON III (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 1555

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Processing

EM software
IDNameVersionCategory
1cryoSPARC3.3.1particle selection
2EPU2.8image acquisition
4cryoSPARC3.3.1CTF correction
9cryoSPARC3.3.1initial Euler assignment
10cryoSPARC3.3.1final Euler assignment
11cryoSPARC3.3.1classification
12cryoSPARC3.3.13D reconstruction
13PHENIX1.20_4459:model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 619090
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 108750 / Algorithm: BACK PROJECTION / Symmetry type: POINT
Atomic model buildingB value: 98 / Protocol: RIGID BODY FIT / Space: REAL
Atomic model buildingChain residue range: 1-419 / Details: own experimental data / Source name: Other / Type: experimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00323310
ELECTRON MICROSCOPYf_angle_d0.6831407
ELECTRON MICROSCOPYf_dihedral_angle_d3.9723159
ELECTRON MICROSCOPYf_chiral_restr0.0423597
ELECTRON MICROSCOPYf_plane_restr0.0044097

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