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- PDB-9ff7: Structure of the BMOE-crosslinked transcription termination facto... -

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Basic information

Entry
Database: PDB / ID: 9ff7
TitleStructure of the BMOE-crosslinked transcription termination factor Rho in the presence of ppGpp; S84C/M405C double mutant
ComponentsTranscription termination factor Rho
KeywordsTRANSCRIPTION / Rho / termination / bacterial stress response / ppGpp
Function / homology
Function and homology information


ATP-dependent activity, acting on RNA / 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 / cytosol
Similarity search - Function
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 ...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
1,1'-ethane-1,2-diylbis(1H-pyrrole-2,5-dione) / Transcription termination factor Rho
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsSaid, N. / Hilal, T. / Wahl, M.C.
Funding support Germany, 1items
OrganizationGrant numberCountry
German Research Foundation (DFG)433623608 Germany
Citation
Journal: Nat Commun / Year: 2025
Title: Nucleotide-induced hyper-oligomerization inactivates transcription termination factor ρ.
Authors: Bing Wang / Nelly Said / Tarek Hilal / Mark Finazzo / Markus C Wahl / Irina Artsimovitch /
Abstract: Bacterial RNA helicase ρ is a genome sentinel that terminates the synthesis of damaged and junk RNAs that are not translated by the ribosome. It is unclear how ρ is regulated during dormancy or ...Bacterial RNA helicase ρ is a genome sentinel that terminates the synthesis of damaged and junk RNAs that are not translated by the ribosome. It is unclear how ρ is regulated during dormancy or stress, when translation is inefficient and RNAs are vulnerable to ρ-mediated release. We use cryogenic electron microscopy, biochemical, and genetic approaches to show that substitutions of residues in the connector between two ρ domains or ADP promote the formation of extended Escherichia coli ρ filaments. By contrast, (p)ppGpp induces the formation of transient ρ dodecamers. Our results demonstrate that ADP and (p)ppGpp nucleotides bound at subunit interfaces inhibit ρ ring closure that underpins the hexamer activation, thus favoring the assembly of inactive higher-order oligomers. Connector substitutions and antibiotics that inhibit RNA and protein syntheses trigger ρ aggregation in the cell. These and other recent data implicate aggregation as a widespread strategy to tune ρ activity.
#1: Journal: bioRxiv / Year: 2023
Title: Transcription termination factor ρ polymerizes under stress.
Authors: Bing Wang / Nelly Said / Tarek Hilal / Mark Finazzo / Markus C Wahl / Irina Artsimovitch /
Abstract: Bacterial RNA helicase ρ is a genome sentinel that terminates synthesis of damaged and junk RNAs that are not translated by the ribosome. Co-transcriptional RNA surveillance by ρ is essential for ...Bacterial RNA helicase ρ is a genome sentinel that terminates synthesis of damaged and junk RNAs that are not translated by the ribosome. Co-transcriptional RNA surveillance by ρ is essential for quality control of the transcriptome during optimal growth. However, it is unclear how bacteria protect their RNAs from overzealous ρ during dormancy or stress, conditions common in natural habitats. Here we used cryogenic electron microscopy, biochemical, and genetic approaches to show that residue substitutions, ADP, or ppGpp promote hyper-oligomerization of ρ. Our results demonstrate that nucleotides bound at subunit interfaces control ρ switching from active hexamers to inactive higher-order oligomers and extended filaments. Polymers formed upon exposure to antibiotics or ppGpp disassemble when stress is relieved, thereby directly linking termination activity to cellular physiology. Inactivation of ρ through hyper-oligomerization is a regulatory strategy shared by RNA polymerases, ribosomes, and metabolic enzymes across all life.
History
DepositionMay 22, 2024Deposition site: PDBE / Processing site: PDBE
Revision 1.0Apr 9, 2025Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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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
G: Transcription termination factor Rho
H: Transcription termination factor Rho
I: Transcription termination factor Rho
J: Transcription termination factor Rho
K: Transcription termination factor Rho
L: Transcription termination factor Rho
hetero molecules


Theoretical massNumber of molelcules
Total (without water)564,85121
Polymers563,65312
Non-polymers1,1989
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area34300 Å2
ΔGint11 kcal/mol
Surface area196610 Å2

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Components

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


Mass: 46971.098 Da / Num. of mol.: 12
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: rho, Z5293, ECs4716 / Production host: Escherichia coli (E. coli)
References: UniProt: P0AG32, Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement
#2: Chemical
ChemComp-ME7 / 1,1'-ethane-1,2-diylbis(1H-pyrrole-2,5-dione)


Mass: 220.182 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: C10H8N2O4
#3: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Mg
Has ligand of interestN
Has protein modificationN

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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: Dimer of transcription termination factor Rho / Type: COMPLEX / Details: S84C/M405C double mutant, BMOE crosslinked / Entity ID: #1 / Source: RECOMBINANT
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 / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / 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 FIELD / Nominal magnification: 96000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 900 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: ZEMLIN TABLEAU
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 40.57 sec. / Electron dose: 44 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON III (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 2218

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Processing

EM software
IDNameVersionCategory
1cryoSPARC4.4particle selection
2PHENIX1.20_4459:model refinement
3EPU2.8image acquisition
5cryoSPARC4.4CTF correction
10cryoSPARC4.4initial Euler assignment
11cryoSPARC4.4final Euler assignment
12cryoSPARC4.4classification
13cryoSPARC4.43D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 840400
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 96116 / Algorithm: BACK PROJECTION / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00240141
ELECTRON MICROSCOPYf_angle_d0.35554060
ELECTRON MICROSCOPYf_dihedral_angle_d4.8235463
ELECTRON MICROSCOPYf_chiral_restr0.0386144
ELECTRON MICROSCOPYf_plane_restr0.0027054

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