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- PDB-9gdb: RNAP-TopoI complex on bubble scaffold - consensus reconstruction -

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Basic information

Entry
Database: PDB / ID: 9gdb
TitleRNAP-TopoI complex on bubble scaffold - consensus reconstruction
Components
  • (DNA-directed RNA polymerase subunit ...) x 4
  • DNA topoisomerase 1
  • RNA
  • non-template DNA strand
  • template DNA strand
KeywordsTRANSCRIPTION / RNA polymerase / topoisomerase / DNA topology
Function / homology
Function and homology information


RNA topoisomerase activity / DNA topoisomerase activity / DNA topoisomerase / DNA topoisomerase type I (single strand cut, ATP-independent) activity / RNA polymerase complex / submerged biofilm formation / cellular response to cell envelope stress / regulation of DNA-templated transcription initiation / DNA topological change / bacterial-type flagellum assembly ...RNA topoisomerase activity / DNA topoisomerase activity / DNA topoisomerase / DNA topoisomerase type I (single strand cut, ATP-independent) activity / RNA polymerase complex / submerged biofilm formation / cellular response to cell envelope stress / regulation of DNA-templated transcription initiation / DNA topological change / bacterial-type flagellum assembly / bacterial-type RNA polymerase core enzyme binding / cytosolic DNA-directed RNA polymerase complex / bacterial-type flagellum-dependent cell motility / nitrate assimilation / regulation of DNA-templated transcription elongation / transcription elongation factor complex / transcription antitermination / cell motility / DNA-templated transcription initiation / ribonucleoside binding / DNA-directed RNA polymerase / DNA-directed RNA polymerase activity / chromosome / response to heat / protein-containing complex assembly / intracellular iron ion homeostasis / protein dimerization activity / response to antibiotic / magnesium ion binding / DNA binding / zinc ion binding / membrane / cytosol / cytoplasm
Similarity search - Function
DNA topoisomerase I, zinc ribbon-like, bacterial-type / : / Topoisomerase I zinc-ribbon-like / Topoisomerase I, zinc finger / DNA topoisomerase, type IA, zn finger / Topoisomerase DNA binding C4 zinc finger / DNA topoisomerase I, bacterial-type / DNA topoisomerase I, type IA / DNA topoisomerase 1, TOPRIM domain / DNA topoisomerase, type IA ...DNA topoisomerase I, zinc ribbon-like, bacterial-type / : / Topoisomerase I zinc-ribbon-like / Topoisomerase I, zinc finger / DNA topoisomerase, type IA, zn finger / Topoisomerase DNA binding C4 zinc finger / DNA topoisomerase I, bacterial-type / DNA topoisomerase I, type IA / DNA topoisomerase 1, TOPRIM domain / DNA topoisomerase, type IA / DNA topoisomerase, type IA, central region, subdomain 2 / DNA topoisomerase, type IA, active site / Topoisomerase (Topo) IA-type active site signature. / Topoisomerase (Topo) IA-type catalytic domain profile. / DNA topoisomerase, type IA, domain 2 / DNA topoisomerase, type IA, DNA-binding domain / DNA topoisomerase, type IA, central / DNA topoisomerase, type IA, central region, subdomain 1 / DNA topoisomerase, type IA, central region, subdomain 3 / DNA topoisomerase, type IA, core domain / DNA topoisomerase / Bacterial DNA topoisomeraes I ATP-binding domain / Bacterial DNA topoisomerase I DNA-binding domain / TOPRIM / Toprim domain / Toprim domain profile. / TOPRIM domain / DNA-directed RNA polymerase, omega subunit / DNA-directed RNA polymerase, subunit beta-prime, bacterial type / DNA-directed RNA polymerase, beta subunit, external 1 domain superfamily / DNA-directed RNA polymerase, beta subunit, external 1 domain / RNA polymerase beta subunit external 1 domain / RNA polymerase, alpha subunit, C-terminal / Bacterial RNA polymerase, alpha chain C terminal domain / DNA-directed RNA polymerase beta subunit, bacterial-type / DNA-directed RNA polymerase, alpha subunit / DNA-directed RNA polymerase, subunit beta-prime / RNA polymerase Rpb6 / RNA polymerase, subunit omega/Rpo6/RPB6 / RNA polymerase Rpb6 / RNA polymerase Rpb2, domain 2 superfamily / RNA polymerase Rpb1, domain 3 superfamily / RPB6/omega subunit-like superfamily / DNA-directed RNA polymerase, insert domain / DNA-directed RNA polymerase, RpoA/D/Rpb3-type / RNA polymerase Rpb3/RpoA insert domain / RNA polymerase Rpb3/Rpb11 dimerisation domain / RNA polymerases D / RNA polymerase Rpb1, clamp domain superfamily / RNA polymerase Rpb1, domain 3 / RNA polymerase Rpb1, domain 3 / RNA polymerase, beta subunit, protrusion / RNA polymerase beta subunit / RNA polymerase Rpb1, domain 1 / RNA polymerase Rpb1, domain 1 / RNA polymerase, alpha subunit / RNA polymerase Rpb1, domain 5 / RNA polymerase Rpb1, domain 4 / RNA polymerase Rpb1, domain 2 / RNA polymerase Rpb1, domain 5 / RNA polymerase Rpb1, domain 4 / DNA-directed RNA polymerase, insert domain superfamily / RNA polymerase Rpb2, domain 2 / RNA polymerase Rpb2, domain 2 / RNA polymerase, N-terminal / RNA polymerase, RBP11-like subunit / RNA polymerase Rpb1, funnel domain superfamily / RNA polymerase I subunit A N-terminus / RNA polymerase, beta subunit, conserved site / RNA polymerase Rpb2, domain 7 / RNA polymerase Rpb2, domain 3 / RNA polymerase Rpb2, OB-fold / RNA polymerase Rpb2, domain 7 / RNA polymerase Rpb2, domain 3 / RNA polymerases beta chain signature. / DNA-directed RNA polymerase, subunit 2, hybrid-binding domain / DNA-directed RNA polymerase, subunit 2 / DNA-directed RNA polymerase, subunit 2, hybrid-binding domain superfamily / RNA polymerase Rpb2, domain 6
Similarity search - Domain/homology
DNA / DNA (> 10) / RNA / RNA (> 10) / DNA topoisomerase 1 / DNA-directed RNA polymerase subunit alpha / DNA-directed RNA polymerase subunit omega / DNA-directed RNA polymerase subunit beta' / DNA-directed RNA polymerase subunit beta
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.8 Å
AuthorsVidmar, V. / Weixlbaumer, A. / Lamour, V.
Funding support France, 2items
OrganizationGrant numberCountry
Agence Nationale de la Recherche (ANR)ANR-21-CE11-0040-01 France
Agence Nationale de la Recherche (ANR)ANR-10-INBS-05 France
CitationJournal: Nat Struct Mol Biol / Year: 2025
Title: DNA topoisomerase I acts as supercoiling sensor for bacterial transcription elongation.
Authors: Vita Vidmar / Céline Borde / Lisa Bruno / Nataliya Miropolskaya / Maria Takacs / Claire Batisse / Charlotte Saint-André / Chengjin Zhu / Olivier Espéli / Valérie Lamour / Albert Weixlbaumer /
Abstract: During transcription, RNA polymerase (RNAP) continuously unwinds and rewinds DNA, generating negative and positive supercoils upstream and downstream, respectively. Using single-particle cryo-EM, we ...During transcription, RNA polymerase (RNAP) continuously unwinds and rewinds DNA, generating negative and positive supercoils upstream and downstream, respectively. Using single-particle cryo-EM, we elucidated how bacterial RNAP and DNA topoisomerase I (TopoI), which relaxes negative supercoils, operate in close spatial proximity. TopoI binds to relaxed DNA upstream of RNAP, and this involves a conformational switch in the TopoI functional domains. This suggests that TopoI exerts a sensing role before the formation of negative supercoils. On DNA substrates mimicking negatively supercoiled DNA, TopoI threads one strand into the active site for cleavage and binds the complementary strand with an auxiliary domain. Transcriptomic and phenotypic analyses suggest that mutations affecting conformational changes in TopoI impact gene expression and operon polarity in bacteria. In summary, we propose a comprehensive model for DNA relaxation in the proximity of active bacterial transcription.
History
DepositionAug 5, 2024Deposition site: PDBE / Processing site: PDBE
Revision 1.0Nov 5, 2025Provider: repository / Type: Initial release
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: DNA-directed RNA polymerase subunit alpha
B: DNA-directed RNA polymerase subunit alpha
C: DNA-directed RNA polymerase subunit beta
D: DNA-directed RNA polymerase subunit beta'
E: DNA-directed RNA polymerase subunit omega
F: DNA topoisomerase 1
N: non-template DNA strand
R: RNA
T: template DNA strand
hetero molecules


Theoretical massNumber of molelcules
Total (without water)528,60115
Polymers528,2509
Non-polymers3516
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

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DNA-directed RNA polymerase subunit ... , 4 types, 5 molecules ABCDE

#1: Protein DNA-directed RNA polymerase subunit alpha / RNAP subunit alpha / RNA polymerase subunit alpha / Transcriptase subunit alpha


Mass: 36558.680 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: rpoA, pez, phs, sez, b3295, JW3257 / Production host: Escherichia coli (E. coli) / References: UniProt: P0A7Z4, DNA-directed RNA polymerase
#2: Protein DNA-directed RNA polymerase subunit beta / RNAP subunit beta / RNA polymerase subunit beta / Transcriptase subunit beta


Mass: 150820.875 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli)
Gene: rpoB, groN, nitB, rif, ron, stl, stv, tabD, b3987, JW3950
Production host: Escherichia coli (E. coli) / References: UniProt: P0A8V2, DNA-directed RNA polymerase
#3: Protein DNA-directed RNA polymerase subunit beta' / RNAP subunit beta' / RNA polymerase subunit beta' / Transcriptase subunit beta'


Mass: 156338.891 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: rpoC, tabB, b3988, JW3951 / Production host: Escherichia coli (E. coli) / References: UniProt: P0A8T7, DNA-directed RNA polymerase
#4: Protein DNA-directed RNA polymerase subunit omega / RNAP omega subunit / RNA polymerase omega subunit / Transcriptase subunit omega


Mass: 10249.547 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: rpoZ, b3649, JW3624 / Production host: Escherichia coli (E. coli) / References: UniProt: P0A800, DNA-directed RNA polymerase

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DNA chain , 2 types, 2 molecules NT

#6: DNA chain non-template DNA strand


Mass: 16387.543 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#8: DNA chain template DNA strand


Mass: 16143.378 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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Protein / RNA chain , 2 types, 2 molecules FR

#5: Protein DNA topoisomerase 1 / DNA topoisomerase I / Omega-protein / Relaxing enzyme / Swivelase / Untwisting enzyme


Mass: 100659.328 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: N-terminal TwinStrep tag / Source: (gene. exp.) Escherichia coli (E. coli) / Gene: topA, supX, b1274, JW1266 / Production host: Escherichia coli (E. coli) / References: UniProt: P06612, DNA topoisomerase
#7: RNA chain RNA


Mass: 4532.764 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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Non-polymers , 2 types, 6 molecules

#9: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg
#10: Chemical
ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: Zn

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Details

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

Component
IDNameTypeEntity IDParent-IDSource
1RNAP-TopoI complex on bubble scaffoldCOMPLEX#1-#80MULTIPLE SOURCES
2DNA-directed RNA polymerase elongation complexCOMPLEX#1-#4, #6-#81RECOMBINANT
3DNA topoisomerase 1COMPLEX#51RECOMBINANT
Molecular weight
IDEntity assembly-IDValue (°)Experimental value
110.52 MDaNO
210.39 MDaNO
310.1 MDaNO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Escherichia coli (E. coli)562
33Escherichia coli (E. coli)562
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
22Escherichia coli (E. coli)562
33Escherichia coli (E. coli)562
Buffer solutionpH: 7.5
Buffer component
IDConc.NameBuffer-ID
110 mMHEPES/KOH1
2150 mMpotassium acetate1
35 mMmagnesium acetate1
40.01 mMzinc chloride1
52 mMDTT1
SpecimenConc.: 10 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: UltrAuFoil 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: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2500 nm / Nominal defocus min: 800 nm
Specimen holderCryogen: NITROGEN
Image recordingElectron dose: 54.03 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

EM software
IDNameVersionCategory
1Warpparticle selection
2SerialEMimage acquisition
4cryoSPARCCTF correction
7UCSF Chimeramodel fitting
8ISOLDEmodel fitting
10PHENIX1.20.1model refinement
11Coot0.9.6model refinement
12cryoSPARCinitial Euler assignment
13cryoSPARCfinal Euler assignment
14cryoSPARCclassification
15cryoSPARC3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 49760 / Symmetry type: POINT
Atomic model buildingProtocol: OTHER / Space: REAL
Details: Initial rigid body fit was done in UCSF Chimera, followed by flexible fitting of TopoI in Isolde and all-atom refinement of RNAP in Phenix and Coot.
Atomic model building
IDPDB-ID 3D fitting-IDAccession codeInitial refinement model-IDSource nameType
16alh

6alh

16alh1PDBexperimental model
24rul

4rul

14rul2PDBexperimental model

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