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- PDB-7ssg: Mfd DNA complex -

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Basic information

Entry
Database: PDB / ID: 7ssg
TitleMfd DNA complex
Components
  • DNA (5'-D(P*TP*GP*GP*CP*GP*GP*CP*GP*AP*GP*GP*C)-3')
  • DNA (5'-D(P*TP*TP*GP*CP*CP*TP*CP*GP*CP*TP*GP*CP*CP*A)-3')
  • Transcription-repair-coupling factor
KeywordsDNA BINDING PROTEIN/DNA / DNA TRANSLOCASE / TRANSCRIPTION-COUPLED DNA REPAIR / HELICASE / ATPASE / DNA BINDING PROTEIN / HYDROLASE-DNA COMPLEX / DNA BINDING PROTEIN-DNA complex
Function / homology
Function and homology information


transcription-coupled nucleotide-excision repair, DNA damage recognition / RNA polymerase core enzyme binding / nucleotide-excision repair, preincision complex assembly / DNA translocase activity / DNA repair complex / transcription-coupled nucleotide-excision repair / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / damaged DNA binding / hydrolase activity / DNA repair ...transcription-coupled nucleotide-excision repair, DNA damage recognition / RNA polymerase core enzyme binding / nucleotide-excision repair, preincision complex assembly / DNA translocase activity / DNA repair complex / transcription-coupled nucleotide-excision repair / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / damaged DNA binding / hydrolase activity / DNA repair / DNA damage response / regulation of DNA-templated transcription / DNA binding / ATP binding / cytosol
Similarity search - Function
: / MFD, D3 domain / : / Transcription-repair coupling factor / Transcription-repair-coupling factor, C-terminal domain / TRCF-like, C-terminal D7 domain / TRCF domain / TRCF / UvrB, interaction domain / UvrB interaction domain ...: / MFD, D3 domain / : / Transcription-repair coupling factor / Transcription-repair-coupling factor, C-terminal domain / TRCF-like, C-terminal D7 domain / TRCF domain / TRCF / UvrB, interaction domain / UvrB interaction domain / CarD-like/TRCF, RNAP-interacting domain / CarD-like/TRCF, RNAP-interacting domain superfamily / CarD-like/TRCF RID domain / CarD-like/TRCF domain / DEAD/DEAH box helicase / DEAD/DEAH box helicase domain / Helicase conserved C-terminal domain / helicase superfamily c-terminal domain / Superfamilies 1 and 2 helicase C-terminal domain profile. / Superfamilies 1 and 2 helicase ATP-binding type-1 domain profile. / DEAD-like helicases superfamily / Helicase, C-terminal / Helicase superfamily 1/2, ATP-binding domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
DNA / DNA (> 10) / Transcription-repair-coupling factor
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 5.2 Å
AuthorsOakley, A.J. / Xu, Z.-Q.
Funding support United States, 2items
OrganizationGrant numberCountry
Australian Research Council (ARC)DP210100365 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)1RM1GM130450 United States
CitationJournal: Nucleic Acids Res / Year: 2022
Title: Mechanism of transcription modulation by the transcription-repair coupling factor.
Authors: Bishnu P Paudel / Zhi-Qiang Xu / Slobodan Jergic / Aaron J Oakley / Nischal Sharma / Simon H J Brown / James C Bouwer / Peter J Lewis / Nicholas E Dixon / Antoine M van Oijen / Harshad Ghodke /
Abstract: Elongation by RNA polymerase is dynamically modulated by accessory factors. The transcription-repair coupling factor (TRCF) recognizes paused/stalled RNAPs and either rescues transcription or ...Elongation by RNA polymerase is dynamically modulated by accessory factors. The transcription-repair coupling factor (TRCF) recognizes paused/stalled RNAPs and either rescues transcription or initiates transcription termination. Precisely how TRCFs choose to execute either outcome remains unclear. With Escherichia coli as a model, we used single-molecule assays to study dynamic modulation of elongation by Mfd, the bacterial TRCF. We found that nucleotide-bound Mfd converts the elongation complex (EC) into a catalytically poised state, presenting the EC with an opportunity to restart transcription. After long-lived residence in this catalytically poised state, ATP hydrolysis by Mfd remodels the EC through an irreversible process leading to loss of the RNA transcript. Further, biophysical studies revealed that the motor domain of Mfd binds and partially melts DNA containing a template strand overhang. The results explain pathway choice determining the fate of the EC and provide a molecular mechanism for transcription modulation by TRCF.
History
DepositionNov 11, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0May 25, 2022Provider: repository / Type: Initial release
Revision 1.1Jul 6, 2022Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID / _citation_author.name
Revision 1.2Jun 5, 2024Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_3d_fitting_list / pdbx_initial_refinement_model
Item: _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id ..._em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Transcription-repair-coupling factor
B: DNA (5'-D(P*TP*TP*GP*CP*CP*TP*CP*GP*CP*TP*GP*CP*CP*A)-3')
C: DNA (5'-D(P*TP*GP*GP*CP*GP*GP*CP*GP*AP*GP*GP*C)-3')


Theoretical massNumber of molelcules
Total (without water)153,3573
Polymers153,3573
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Transcription-repair-coupling factor / TRCF


Mass: 130152.422 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Strain: K12 / Gene: mfd, b1114, JW1100 / Production host: Escherichia coli BL21 (bacteria) / Variant (production host): recA
References: UniProt: P30958, Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement
#2: DNA chain DNA (5'-D(P*TP*TP*GP*CP*CP*TP*CP*GP*CP*TP*GP*CP*CP*A)-3')


Mass: 17597.205 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Escherichia coli (E. coli)
#3: DNA chain DNA (5'-D(P*TP*GP*GP*CP*GP*GP*CP*GP*AP*GP*GP*C)-3')


Mass: 5607.617 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Escherichia coli (E. coli)

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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: mfd complex with DNA / Type: COMPLEX / Entity ID: all / Source: MULTIPLE SOURCES
Molecular weightValue: 0.130 MDa / Experimental value: YES
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMTris/HCl1
240 mMpotassium chlorideKCl1
35 mMmagnesium chlorideMgCl21
41 mMbeta-mercaptoethanol1
SpecimenConc.: 1.6 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: ADP, NaF and AlCl3 were added to final concentrations of 2, 5 and 0.5 mM, respectively.
Specimen supportGrid material: GOLD / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 279 K

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Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TALOS ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 1900 nm / Nominal defocus min: 1000 nm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 50 sec. / Electron dose: 50 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON III (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 472

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Processing

SoftwareName: PHENIX / Version: 1.19.1_4122: / Classification: refinement
EM software
IDNameVersionCategory
1cryoSPARC2particle selection
2EPUimage acquisition
7Coot0.9.6model fitting
11cryoSPARC2classification
12cryoSPARC23D reconstruction
13PHENIX1.2model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 110562
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 5.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 31718 / Algorithm: BACK PROJECTION / Num. of class averages: 3 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL / Target criteria: Correlation coeficient
Atomic model buildingPDB-ID: 6XEO

6xeo


Accession code: 6XEO / Source name: PDB / Type: experimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0024141
ELECTRON MICROSCOPYf_angle_d0.4565798
ELECTRON MICROSCOPYf_dihedral_angle_d20.631613
ELECTRON MICROSCOPYf_chiral_restr0.035659
ELECTRON MICROSCOPYf_plane_restr0.004591

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