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Basic information

Entry
Database: PDB / ID: 9kev
TitleCryo-EM structure of Mycobacterium tuberculosis transcription activation complex with six PhoP molecules (composite map)
Components
  • (DNA-directed RNA polymerase subunit ...) x 4
  • Non-template strand DNA of the promoter
  • Possible two component system response transcriptional positive regulator PhoP
  • RNA polymerase sigma factor SigA
  • Template strand DNA of the promoter
KeywordsGENE REGULATION/DNA / bacterial RNA polymerase / GENE REGULATION-DNA COMPLEX
Function / homology
Function and homology information


protein secretion by the type VII secretion system / glycolipid biosynthetic process / response to water / Antimicrobial action and antimicrobial resistance in Mtb / phosphorelay response regulator activity / sigma factor activity / bacterial-type RNA polymerase core enzyme binding / cytosolic DNA-directed RNA polymerase complex / positive regulation of lipid biosynthetic process / DNA-directed RNA polymerase complex ...protein secretion by the type VII secretion system / glycolipid biosynthetic process / response to water / Antimicrobial action and antimicrobial resistance in Mtb / phosphorelay response regulator activity / sigma factor activity / bacterial-type RNA polymerase core enzyme binding / cytosolic DNA-directed RNA polymerase complex / positive regulation of lipid biosynthetic process / DNA-directed RNA polymerase complex / peptidoglycan-based cell wall / DNA-templated transcription initiation / protein-DNA complex / ribonucleoside binding / : / : / : / : / : / : / DNA-directed RNA polymerase / response to oxidative stress / transcription cis-regulatory region binding / protein dimerization activity / response to antibiotic / negative regulation of DNA-templated transcription / positive regulation of gene expression / regulation of DNA-templated transcription / positive regulation of DNA-templated transcription / magnesium ion binding / DNA binding / zinc ion binding / metal ion binding / plasma membrane / cytosol / cytoplasm
Similarity search - Function
OmpR/PhoB-type DNA-binding domain / Transcriptional regulatory protein, C terminal / OmpR/PhoB-type DNA-binding domain profile. / Transcriptional regulatory protein, C terminal / Transcriptional regulatory protein WalR-like / Signal transduction response regulator, C-terminal effector / Sigma-70 factors family signature 1. / RNA polymerase sigma factor RpoD, C-terminal / RNA polymerase sigma factor RpoD / : ...OmpR/PhoB-type DNA-binding domain / Transcriptional regulatory protein, C terminal / OmpR/PhoB-type DNA-binding domain profile. / Transcriptional regulatory protein, C terminal / Transcriptional regulatory protein WalR-like / Signal transduction response regulator, C-terminal effector / Sigma-70 factors family signature 1. / RNA polymerase sigma factor RpoD, C-terminal / RNA polymerase sigma factor RpoD / : / RNA polymerase sigma-70 region 1.2 / Sigma-70 factor, region 1.2 / RNA polymerase sigma-70 region 3 / Sigma-70 region 3 / Sigma-70 factors family signature 2. / RNA polymerase sigma-70 / RNA polymerase sigma-70 region 4 / Sigma-70, region 4 / RNA polymerase sigma-70 region 2 / RNA polymerase sigma-70 like domain / Sigma-70 region 2 / RNA polymerase sigma factor, region 2 / RNA polymerase sigma factor, region 3/4-like / Response regulator receiver domain / cheY-homologous receiver domain / Signal transduction response regulator, receiver domain / Response regulatory domain profile. / CheY-like superfamily / 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, alpha subunit / DNA-directed RNA polymerase beta subunit, bacterial-type / 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 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 Rpb1, funnel domain superfamily / RNA polymerase I subunit A N-terminus / RNA polymerase, RBP11-like subunit / RNA polymerase Rpb2, domain 2 / RNA polymerase Rpb2, domain 2 / 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 / Winged helix-like DNA-binding domain superfamily
Similarity search - Domain/homology
DNA / DNA (> 10) / DNA (> 100) / Possible two component system response transcriptional positive regulator PhoP / RNA polymerase sigma factor SigA / DNA-directed RNA polymerase subunit omega / DNA-directed RNA polymerase subunit beta' / DNA-directed RNA polymerase subunit beta / DNA-directed RNA polymerase subunit alpha
Similarity search - Component
Biological speciesMycobacterium tuberculosis H37Rv (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.31 Å
AuthorsLin, W. / Feng, Y.
Funding support China, 1items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC) China
CitationJournal: Nat Commun / Year: 2025
Title: Structural insights into transcription regulation of the global OmpR/PhoB family regulator PhoP from Mycobacterium tuberculosis.
Authors: Jing Shi / Zhenzhen Feng / Qian Song / Aijia Wen / Tianyu Liu / Liqiao Xu / Zonghang Ye / Simin Xu / Fei Gao / Liuxiang Xiao / Jiapeng Zhu / Kalyan Das / Guoping Zhao / Jie Li / Yu Feng / Wei Lin /
Abstract: As a global transcription activator or repressor, the representative OmpR/PhoB family response regulator PhoP plays a crucial role in regulating bacterial pathogenicity and stress adaptation. ...As a global transcription activator or repressor, the representative OmpR/PhoB family response regulator PhoP plays a crucial role in regulating bacterial pathogenicity and stress adaptation. However, the molecular mechanisms underlying the transcriptional regulation that define its differential functions remain largely unclear. In the present study, we determine three cryo-EM structures of Mycobacterium tuberculosis (Mtb) PhoP-dependent transcription activation complexes (PhoP-TACs) and build one preliminary cryo-EM structure model of Mtb PhoP-dependent transcription repression complex (PhoP-TRC). In PhoP-TACs, tandem PhoP dimers cooperatively recognize various types of promoters through conserved PhoP-PHO box interactions, which displace the canonical interactions between the -35 element and σR4 of RNA polymerase (RNAP), unraveling complex transcription activation mechanisms of PhoP. In PhoP-TRC, one PhoP dimer binds and significantly distorts the upstream PHO box of the promoter cross-talked with the global nitrogen regulator GlnR through the PhoP-PHO box, PhoP-GlnR and αCTD-DNA interactions. This unique binding of PhoP creates steric hindrances that prevent additional GlnR binding, positioning PhoP within a unique 'competitive occluding model', as supported by prior biochemical observations. Collectively, these findings reveal the dual molecular mechanisms of PhoP-dependent transcription regulation, and offer valuable insights for further exploration of the enormous PhoP-like OmpR/PhoB family response regulators.
History
DepositionNov 5, 2024Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Mar 5, 2025Provider: repository / Type: Initial release
Revision 1.0Mar 5, 2025Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Mar 5, 2025Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Mar 5, 2025Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release
Revision 1.1Mar 12, 2025Group: Data collection / Database references / Category: citation / em_admin
Item: _citation.page_last / _citation.pdbx_database_id_PubMed ..._citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _em_admin.last_update
Revision 1.1Mar 12, 2025Data content type: EM metadata / Data content type: EM metadata / EM metadata / Group: Database references / Experimental summary / Data content type: EM metadata / EM metadata / Category: citation / em_admin
Data content type: EM metadata / EM metadata ...EM metadata / EM metadata / EM metadata / EM metadata
Item: _citation.page_last / _citation.pdbx_database_id_PubMed ..._citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _em_admin.last_update

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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
H: Non-template strand DNA of the promoter
G: Template strand DNA of the promoter
F: RNA polymerase sigma factor SigA
J: Possible two component system response transcriptional positive regulator PhoP
K: Possible two component system response transcriptional positive regulator PhoP
M: Possible two component system response transcriptional positive regulator PhoP
L: Possible two component system response transcriptional positive regulator PhoP
N: Possible two component system response transcriptional positive regulator PhoP
O: Possible two component system response transcriptional positive regulator PhoP
hetero molecules


Theoretical massNumber of molelcules
Total (without water)654,48617
Polymers654,33114
Non-polymers1553
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: 37745.328 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycobacterium tuberculosis H37Rv (bacteria)
Gene: rpoA / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / References: UniProt: P9WGZ1, DNA-directed RNA polymerase
#2: Protein DNA-directed RNA polymerase subunit beta / RNAP subunit beta / RNA polymerase subunit beta / Transcriptase subunit beta


Mass: 130018.828 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycobacterium tuberculosis H37Rv (bacteria)
Gene: rpoB / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / References: UniProt: P9WGY9, DNA-directed RNA polymerase
#3: Protein DNA-directed RNA polymerase subunit beta' / RNAP subunit beta' / RNA polymerase subunit beta' / Transcriptase subunit beta'


Mass: 146968.969 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycobacterium tuberculosis H37Rv (bacteria)
Gene: rpoC / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / References: UniProt: P9WGY7, DNA-directed RNA polymerase
#4: Protein DNA-directed RNA polymerase subunit omega / RNAP omega subunit / RNA polymerase omega subunit / Transcriptase subunit omega


Mass: 11851.140 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycobacterium tuberculosis H37Rv (bacteria)
Gene: rpoZ / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / References: UniProt: P9WGY5, DNA-directed RNA polymerase

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DNA chain , 2 types, 2 molecules HG

#5: DNA chain Non-template strand DNA of the promoter


Mass: 33464.332 Da / Num. of mol.: 1 / Source method: obtained synthetically
Source: (synth.) Mycobacterium tuberculosis H37Rv (bacteria)
#6: DNA chain Template strand DNA of the promoter


Mass: 33357.250 Da / Num. of mol.: 1 / Source method: obtained synthetically
Source: (synth.) Mycobacterium tuberculosis H37Rv (bacteria)

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Protein , 2 types, 7 molecules FJKMLNO

#7: Protein RNA polymerase sigma factor SigA / Sigma-A


Mass: 57877.160 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycobacterium tuberculosis H37Rv (bacteria)
Gene: sigA / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / References: UniProt: P9WGI1
#8: Protein
Possible two component system response transcriptional positive regulator PhoP


Mass: 27550.488 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycobacterium tuberculosis H37Rv (bacteria)
Gene: phoP / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / References: UniProt: P71814

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Non-polymers , 2 types, 3 molecules

#9: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Zn / Feature type: SUBJECT OF INVESTIGATION
#10: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION

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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: Mycobacterium tuberculosis transcription activation complex with six PhoP molecules (composite map)
Type: COMPLEX / Entity ID: #1-#8 / Source: RECOMBINANT
Source (natural)Organism: Mycobacterium tuberculosis (bacteria)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 7.9
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: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2000 nm / Nominal defocus min: 1000 nm
Image recordingElectron dose: 52 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

EM softwareName: PHENIX / Version: 1.20.1_4487: / Category: model refinement
CTF correctionType: NONE
3D reconstructionResolution: 3.31 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 227755 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00335450
ELECTRON MICROSCOPYf_angle_d0.60148922
ELECTRON MICROSCOPYf_dihedral_angle_d21.6736286
ELECTRON MICROSCOPYf_chiral_restr0.0415591
ELECTRON MICROSCOPYf_plane_restr0.0055667

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