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- PDB-7kif: Mycobacterium tuberculosis WT RNAP transcription open promoter co... -

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Basic information

Entry
Database: PDB / ID: 7kif
TitleMycobacterium tuberculosis WT RNAP transcription open promoter complex with WhiB7 transcription factor
Components
  • (DNA-directed RNA polymerase subunit ...Polymerase) x 4
  • (RNA polymerase-binding ...) x 2
  • DNA (55-MER)
  • DNA (63-MER)
  • Probable transcriptional regulator WhiB7
  • RNA polymerase sigma factor SigA
KeywordsTRANSCRIPTION / TRANSFERASE/DNA / RNA polymerase / Transcription factor / Mycobacterium tuberculosis / Iron cluster / TRANSFERASE-DNA complex
Function / homology
Function and homology information


dinitrosyl-iron complex binding / protein-disulfide reductase (NAD(P)H) activity / bacterial-type RNA polymerase core enzyme binding / response to fatty acid / sigma factor activity / cell redox homeostasis / DNA-directed RNA polymerase complex / DNA-templated transcription initiation / ribonucleoside binding / DNA-directed 5'-3' RNA polymerase activity ...dinitrosyl-iron complex binding / protein-disulfide reductase (NAD(P)H) activity / bacterial-type RNA polymerase core enzyme binding / response to fatty acid / sigma factor activity / cell redox homeostasis / DNA-directed RNA polymerase complex / DNA-templated transcription initiation / ribonucleoside binding / DNA-directed 5'-3' RNA polymerase activity / DNA-directed RNA polymerase / 4 iron, 4 sulfur cluster binding / protein dimerization activity / response to antibiotic / negative regulation of DNA-templated transcription / DNA-templated transcription / positive regulation of DNA-templated transcription / magnesium ion binding / DNA binding / zinc ion binding / metal ion binding / cytoplasm
Similarity search - Function
AT hook motif / Transcription factor WhiB / WhiB-like iron-sulfur binding domain / Transcription factor WhiB / 4Fe-4S WhiB-like (Wbl)-type iron-sulfur binding domain profile. / : / CarD, C-terminal / CarD-like, C-terminal domain / RNA polymerase-binding protein RbpA / RbpA superfamily ...AT hook motif / Transcription factor WhiB / WhiB-like iron-sulfur binding domain / Transcription factor WhiB / 4Fe-4S WhiB-like (Wbl)-type iron-sulfur binding domain profile. / : / CarD, C-terminal / CarD-like, C-terminal domain / RNA polymerase-binding protein RbpA / RbpA superfamily / RNA polymerase-binding protein / CarD-like/TRCF, RNAP-interacting domain / CarD-like/TRCF, RNAP-interacting domain superfamily / CarD-like/TRCF RID domain / CarD-like/TRCF domain / AT hook, DNA-binding motif / 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 / 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 / RNA polymerase Rpb6 / RNA polymerase, subunit omega/Rpo6/RPB6 / RNA polymerase Rpb6 / RNA polymerase Rpb1, domain 3 superfamily / RNA polymerase Rpb1, clamp domain superfamily / RPB6/omega subunit-like superfamily / DNA-directed RNA polymerase, subunit beta-prime / RNA polymerase Rpb1, domain 3 / RNA polymerase Rpb1, domain 3 / RNA polymerase Rpb2, domain 2 superfamily / RNA polymerase Rpb1, domain 1 / RNA polymerase Rpb1, domain 1 / RNA polymerase, alpha subunit / RNA polymerase Rpb1, domain 4 / RNA polymerase Rpb1, domain 2 / RNA polymerase Rpb1, domain 4 / RNA polymerase, N-terminal / RNA polymerase Rpb1, funnel domain superfamily / RNA polymerase I subunit A N-terminus / RNA polymerase Rpb1, domain 5 / RNA polymerase Rpb1, domain 5 / RNA polymerase, beta subunit, protrusion / RNA polymerase beta subunit / 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 / DNA-directed RNA polymerase, insert domain superfamily / 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
IRON/SULFUR CLUSTER / DNA / DNA (> 10) / DNA (> 100) / DNA-directed RNA polymerase subunit beta' / RNA polymerase sigma factor SigA / DNA-directed RNA polymerase subunit omega / DNA-directed RNA polymerase subunit beta / DNA-directed RNA polymerase subunit alpha / RNA polymerase-binding protein RbpA ...IRON/SULFUR CLUSTER / DNA / DNA (> 10) / DNA (> 100) / DNA-directed RNA polymerase subunit beta' / RNA polymerase sigma factor SigA / DNA-directed RNA polymerase subunit omega / DNA-directed RNA polymerase subunit beta / DNA-directed RNA polymerase subunit alpha / RNA polymerase-binding protein RbpA / RNA polymerase-binding transcription factor CarD / Probable transcriptional regulator WhiB7
Similarity search - Component
Biological speciesMycobacterium tuberculosis (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.94 Å
AuthorsLilic, M. / Darst, S.A. / Campbell, E.A.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS) United States
CitationJournal: Mol Cell / Year: 2021
Title: Structural basis of transcriptional activation by the Mycobacterium tuberculosis intrinsic antibiotic-resistance transcription factor WhiB7.
Authors: Mirjana Lilic / Seth A Darst / Elizabeth A Campbell /
Abstract: In pathogenic mycobacteria, transcriptional responses to antibiotics result in induced antibiotic resistance. WhiB7 belongs to the Actinobacteria-specific family of Fe-S-containing transcription ...In pathogenic mycobacteria, transcriptional responses to antibiotics result in induced antibiotic resistance. WhiB7 belongs to the Actinobacteria-specific family of Fe-S-containing transcription factors and plays a crucial role in inducible antibiotic resistance in mycobacteria. Here, we present cryoelectron microscopy structures of Mycobacterium tuberculosis transcriptional regulatory complexes comprising RNA polymerase σ-holoenzyme, global regulators CarD and RbpA, and WhiB7, bound to a WhiB7-regulated promoter. The structures reveal how WhiB7 interacts with σ-holoenzyme while simultaneously interacting with an AT-rich sequence element via its AT-hook. Evidently, AT-hooks, rare elements in bacteria yet prevalent in eukaryotes, bind to target AT-rich DNA sequences similarly to the nuclear chromosome binding proteins. Unexpectedly, a subset of particles contained a WhiB7-stabilized closed promoter complex, revealing this intermediate's structure, and we apply kinetic modeling and biochemical assays to rationalize how WhiB7 activates transcription. Altogether, our work presents a comprehensive view of how WhiB7 serves to activate gene expression leading to antibiotic resistance.
History
DepositionOct 23, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Apr 21, 2021Provider: repository / Type: Initial release
Revision 1.1Nov 3, 2021Group: Database references / Category: citation / citation_author / database_2
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 / _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

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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: RNA polymerase sigma factor SigA
J: RNA polymerase-binding protein RbpA
M: RNA polymerase-binding transcription factor CarD
O: DNA (63-MER)
P: DNA (55-MER)
Z: Probable transcriptional regulator WhiB7
hetero molecules


Theoretical massNumber of molelcules
Total (without water)524,91915
Polymers524,41211
Non-polymers5074
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area63030 Å2
ΔGint-367 kcal/mol
Surface area168840 Å2

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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 / Polymerase / 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 (bacteria) / Gene: rpoA / Production host: Escherichia coli (E. coli) / References: UniProt: A5U8D3, DNA-directed RNA polymerase
#2: Protein DNA-directed RNA polymerase subunit beta / Polymerase / RNAP subunit beta / RNA polymerase subunit beta / Transcriptase subunit beta


Mass: 129372.031 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycobacterium tuberculosis (bacteria) / Gene: rpoB, MRA_0676 / Production host: Escherichia coli (E. coli) / References: UniProt: A5U052, DNA-directed RNA polymerase
#3: Protein DNA-directed RNA polymerase subunit beta' / Polymerase / RNAP subunit beta' / RNA polymerase subunit beta' / Transcriptase subunit beta'


Mass: 147097.094 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycobacterium tuberculosis (bacteria)
Gene: rpoC, E5M52_05480, ERS007665_00591, ERS013471_00574, ERS023446_00410, ERS075361_00813, ERS094182_01340, F6W99_03342
Production host: Escherichia coli (E. coli)
References: UniProt: A0A045J9E2, DNA-directed RNA polymerase
#4: Protein DNA-directed RNA polymerase subunit omega / Polymerase / RNAP omega subunit / RNA polymerase omega subunit / Transcriptase subunit omega


Mass: 11819.075 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycobacterium tuberculosis (bacteria)
Gene: rpoZ, ERS007703_04032, ERS007720_04749, ERS094182_01030
Production host: Escherichia coli (E. coli)
References: UniProt: A0A0T9N9K3, DNA-directed RNA polymerase

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Protein , 2 types, 2 molecules FZ

#5: Protein RNA polymerase sigma factor SigA


Mass: 57845.098 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycobacterium tuberculosis (bacteria) / Gene: sigA, ERDMAN_2966 / Production host: Escherichia coli (E. coli) / References: UniProt: A0A0H3LGM9
#10: Protein Probable transcriptional regulator WhiB7


Mass: 10153.804 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycobacterium tuberculosis (bacteria) / Gene: whiB7, Rv3197A, Rv3197.1 / Production host: Escherichia coli (E. coli) / References: UniProt: Q6MX01

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RNA polymerase-binding ... , 2 types, 2 molecules JM

#6: Protein RNA polymerase-binding protein RbpA


Mass: 12993.695 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycobacterium tuberculosis (bacteria) / Gene: rbpA / Production host: Escherichia coli (E. coli) / References: UniProt: P9WHJ4
#7: Protein RNA polymerase-binding transcription factor CarD


Mass: 17933.361 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycobacterium tuberculosis (bacteria) / Gene: carD / Production host: Escherichia coli (E. coli) / References: UniProt: P9WJG2

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DNA chain , 2 types, 2 molecules OP

#8: DNA chain DNA (63-MER)


Mass: 30844.654 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Mycobacterium tuberculosis (bacteria)
#9: DNA chain DNA (55-MER)


Mass: 30862.684 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Mycobacterium tuberculosis (bacteria)

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Non-polymers , 3 types, 4 molecules

#11: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Zn
#12: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg
#13: Chemical ChemComp-SF4 / IRON/SULFUR CLUSTER / Iron–sulfur cluster


Mass: 351.640 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Fe4S4 / Feature type: SUBJECT OF INVESTIGATION

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Details

Has ligand of interestY

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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 WT RNAP transcription open promoter complex with WhiB7 transcription factor
Type: COMPLEX / Entity ID: #1-#10 / Source: MULTIPLE SOURCES
Source (natural)Organism: Mycobacterium tuberculosis (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
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 FIELDBright-field microscopy
Image recordingElectron dose: 66 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
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.94 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 87743 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00931082
ELECTRON MICROSCOPYf_angle_d0.85542606
ELECTRON MICROSCOPYf_dihedral_angle_d21.85811961
ELECTRON MICROSCOPYf_chiral_restr0.0554847
ELECTRON MICROSCOPYf_plane_restr0.0075188

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