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- PDB-6x2f: Mfd-bound E.coli RNA polymerase elongation complex - L2 state -

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Basic information

Entry
Database: PDB / ID: 6x2f
TitleMfd-bound E.coli RNA polymerase elongation complex - L2 state
Components
  • (DNA (64-MER)) x 2
  • (DNA-directed RNA polymerase subunit ...) x 4
  • RNA (20-MER)
  • Transcription-repair-coupling factor
KeywordsTRANSCRIPTION/RNA/DNA / Transcription-coupled DNA repair / DNA translocase / elongation complex / RNA polymerase / TRANSCRIPTION / TRANSCRIPTION-RNA-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 / RNA polymerase complex / submerged biofilm formation / cellular response to cell envelope stress / regulation of DNA-templated transcription initiation / bacterial-type flagellum assembly ...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 / RNA polymerase complex / submerged biofilm formation / cellular response to cell envelope stress / regulation of DNA-templated transcription initiation / 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 / transcription-coupled nucleotide-excision repair / DNA-directed RNA polymerase complex / DNA helicase activity / regulation of DNA-templated transcription elongation / transcription elongation factor complex / transcription antitermination / cell motility / DNA-templated transcription initiation / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / ribonucleoside binding / DNA-directed RNA polymerase / DNA-directed RNA polymerase activity / response to heat / protein-containing complex assembly / damaged DNA binding / intracellular iron ion homeostasis / hydrolase activity / protein dimerization activity / response to antibiotic / DNA repair / DNA-templated transcription / DNA damage response / regulation of DNA-templated transcription / magnesium ion binding / DNA binding / zinc ion binding / ATP binding / membrane / cytoplasm / cytosol
Similarity search - Function
Transcription-repair-coupling factor, D7 domain / Penicillin-binding protein 1b fold / Penicillin-binding protein 1b domain / : / MFD, D3 domain / Transcription-repair coupling factor / Transcription-repair-coupling factor, C-terminal domain / TRCF-like, C-terminal D7 domain / TRCF domain / TRCF ...Transcription-repair-coupling factor, D7 domain / Penicillin-binding protein 1b fold / Penicillin-binding protein 1b 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 / RNA polymerase Rpb1 funnel domain / Topoisomerase I; Chain A, domain 4 / 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 / Aspartate Aminotransferase, domain 1 / DEAD/DEAH box helicase domain / DEAD/DEAH box helicase / DNA-directed RNA polymerase, subunit beta-prime / RNA polymerase Rpb6 / Helicase conserved C-terminal domain / 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 Rpb1, domain 1 / RNA polymerase Rpb1, domain 1 / RNA polymerase, beta subunit, protrusion / RNA polymerase beta subunit / 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, N-terminal / RNA polymerase Rpb2, domain 2 / RNA polymerase Rpb1, funnel domain superfamily / RNA polymerase Rpb2, domain 2 / RNA polymerase I subunit A N-terminus / RNA polymerase, RBP11-like subunit / 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 / 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 nucleotide triphosphate hydrolases / Alpha-Beta Complex / Rossmann fold / P-loop containing nucleoside triphosphate hydrolase / 2-Layer Sandwich / Orthogonal Bundle / 3-Layer(aba) Sandwich / Mainly Alpha / Alpha Beta
Similarity search - Domain/homology
ADENOSINE-5'-DIPHOSPHATE / DNA / DNA (> 10) / RNA / RNA (> 10) / Transcription-repair-coupling factor / DNA-directed RNA polymerase subunit alpha / DNA-directed RNA polymerase subunit beta' / DNA-directed RNA polymerase subunit alpha / DNA-directed RNA polymerase subunit omega ...ADENOSINE-5'-DIPHOSPHATE / DNA / DNA (> 10) / RNA / RNA (> 10) / Transcription-repair-coupling factor / DNA-directed RNA polymerase subunit alpha / DNA-directed RNA polymerase subunit beta' / DNA-directed RNA polymerase subunit alpha / DNA-directed RNA polymerase subunit omega / DNA-directed RNA polymerase subunit omega / DNA-directed RNA polymerase subunit beta' / DNA-directed RNA polymerase subunit beta / DNA-directed RNA polymerase subunit beta / Transcription-repair-coupling factor
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4 Å
AuthorsLlewellyn, E. / Chen, J. / Kang, J.Y. / Darst, S.A.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35 GM118130 United States
CitationJournal: Elife / Year: 2021
Title: Structural basis for transcription complex disruption by the Mfd translocase.
Authors: Jin Young Kang / Eliza Llewellyn / James Chen / Paul Dominic B Olinares / Joshua Brewer / Brian T Chait / Elizabeth A Campbell / Seth A Darst /
Abstract: Transcription-coupled repair (TCR) is a sub-pathway of nucleotide excision repair (NER) that preferentially removes lesions from the template-strand (t-strand) that stall RNA polymerase (RNAP) ...Transcription-coupled repair (TCR) is a sub-pathway of nucleotide excision repair (NER) that preferentially removes lesions from the template-strand (t-strand) that stall RNA polymerase (RNAP) elongation complexes (ECs). Mfd mediates TCR in bacteria by removing the stalled RNAP concealing the lesion and recruiting Uvr(A)BC. We used cryo-electron microscopy to visualize Mfd engaging with a stalled EC and attempting to dislodge the RNAP. We visualized seven distinct Mfd-EC complexes in both ATP and ADP-bound states. The structures explain how Mfd is remodeled from its repressed conformation, how the UvrA-interacting surface of Mfd is hidden during most of the remodeling process to prevent premature engagement with the NER pathway, how Mfd alters the RNAP conformation to facilitate disassembly, and how Mfd forms a processive translocation complex after dislodging the RNAP. Our results reveal an elaborate mechanism for how Mfd kinetically discriminates paused from stalled ECs and disassembles stalled ECs to initiate TCR.
History
DepositionMay 20, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Feb 3, 2021Provider: repository / Type: Initial release
Revision 1.0Feb 3, 2021Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Feb 3, 2021Data content type: Additional map / Data content type: Additional map / Provider: repository / Type: Initial release
Revision 1.0Feb 3, 2021Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Feb 3, 2021Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release
Revision 1.0Feb 3, 2021Data content type: Additional map / Data content type: Additional map / Provider: repository / Type: Initial release
Revision 1.0Feb 3, 2021Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Feb 3, 2021Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release
Revision 1.1Mar 6, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession
Revision 1.2May 14, 2025Group: Data collection / Structure summary / Category: em_admin / em_software / pdbx_entry_details
Item: _em_admin.last_update / _em_software.name / _pdbx_entry_details.has_protein_modification
Revision 1.1May 14, 2025Data content type: EM metadata / Data content type: EM metadata / EM metadata / Group: Data processing / Experimental summary / Data content type: EM metadata / EM metadata / Category: em_admin / em_software / Data content type: EM metadata / EM metadata / Item: _em_admin.last_update / _em_software.name

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Structure visualization

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Assembly

Deposited unit
A: Transcription-repair-coupling factor
G: DNA-directed RNA polymerase subunit alpha
H: DNA-directed RNA polymerase subunit alpha
I: DNA-directed RNA polymerase subunit beta
J: DNA-directed RNA polymerase subunit beta'
K: DNA-directed RNA polymerase subunit omega
R: RNA (20-MER)
P: DNA (64-MER)
Q: DNA (64-MER)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)566,48413
Polymers565,9019
Non-polymers5824
Water00
1


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

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Components

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

#2: 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 / Production host: Escherichia coli BL21(DE3) (bacteria)
References: UniProt: A0A073G207, UniProt: P0A7Z4*PLUS, DNA-directed RNA polymerase
#3: 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, Z5560, ECs4910 / Production host: Escherichia coli BL21(DE3) (bacteria)
References: UniProt: P0A8V4, UniProt: P0A8V2*PLUS, DNA-directed RNA polymerase
#4: Protein DNA-directed RNA polymerase subunit beta' / RNAP subunit beta' / RNA polymerase subunit beta' / Transcriptase subunit beta'


Mass: 155366.781 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: rpoC, BvCmsKKP036_03580 / Production host: Escherichia coli BL21(DE3) (bacteria)
References: UniProt: A0A4S1NBU2, UniProt: P0A8T7*PLUS, DNA-directed RNA polymerase
#5: 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, Z5075, ECs4524 / Production host: Escherichia coli BL21(DE3) (bacteria)
References: UniProt: P0A802, UniProt: P0A800*PLUS, DNA-directed RNA polymerase

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DNA chain , 2 types, 2 molecules PQ

#7: DNA chain DNA (64-MER)


Mass: 19630.492 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Escherichia coli (E. coli)
#8: DNA chain DNA (64-MER)


Mass: 19748.631 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Escherichia coli (E. coli)

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Protein / RNA chain , 2 types, 2 molecules AR

#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)
Gene: mfd, ACU57_04350, AUQ13_23175, BW690_23090, BZL31_08375, C5N07_18685, C9Z39_13430, CA593_22910, CI694_24470, CIG45_16475, D0X26_19775, D2185_03780, DAH34_08070, DBQ99_15575, E2119_09560, E4K55_ ...Gene: mfd, ACU57_04350, AUQ13_23175, BW690_23090, BZL31_08375, C5N07_18685, C9Z39_13430, CA593_22910, CI694_24470, CIG45_16475, D0X26_19775, D2185_03780, DAH34_08070, DBQ99_15575, E2119_09560, E4K55_21680, EAI52_08950, EEP23_04080, EI021_06895, EI028_16020, EIZ93_13025, EPT01_12285, EXX71_16985, EYD11_13865, F1E19_06650, FV293_24105, FWK02_07210, PGD_02181
Production host: Escherichia coli BL21(DE3) (bacteria)
References: UniProt: A0A024L3Y3, UniProt: P30958*PLUS, Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement
#6: RNA chain RNA (20-MER)


Mass: 6815.149 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Escherichia coli (E. coli)

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Non-polymers , 3 types, 4 molecules

#9: Chemical ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE


Mass: 427.201 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Feature type: SUBJECT OF INVESTIGATION / Comment: ADP, energy-carrying molecule*YM
#10: Chemical ChemComp-MG / MAGNESIUM ION


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


Mass: 65.409 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Zn

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Details

Has ligand of interestY
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: Escherichia coli Mfd-RNAP elongation complex: L2 state
Type: COMPLEX / Entity ID: #1-#8 / Source: RECOMBINANT
Molecular weightValue: 0.57 MDa / Experimental value: NO
Source (natural)Organism: Escherichia coli (E. coli)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 8
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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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
Image recordingElectron dose: 60 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.18.2_3874: / Classification: refinement
EM softwareName: PHENIX / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 16000 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00836878
ELECTRON MICROSCOPYf_angle_d1.13850363
ELECTRON MICROSCOPYf_dihedral_angle_d16.4685868
ELECTRON MICROSCOPYf_chiral_restr0.0645744
ELECTRON MICROSCOPYf_plane_restr0.0076195

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