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- PDB-8dy9: Streptomyces venezuelae RNAP unconstrained open promoter complex ... -

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Basic information

Entry
Database: PDB / ID: 8dy9
TitleStreptomyces venezuelae RNAP unconstrained open promoter complex with WhiA and WhiB transcription factors
Components
  • (DNA (38-MER)) x 2
  • (DNA-directed RNA polymerase subunit ...Polymerase) x 4
  • Probable cell division protein WhiA
  • RNA polymerase sigma factor SigA
  • RNA polymerase-binding protein RbpA
  • Transcriptional regulator WhiB
KeywordsTRANSCRIPTION/TRANSFERASE/DNA / RNA polymerase / Transcription factor / Iron cluster / TRANSCRIPTION-TRANSFERASE-DNA complex
Function / homology
Function and homology information


regulation of sporulation / dinitrosyl-iron complex binding / bacterial-type RNA polymerase core enzyme binding / sigma factor activity / 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 ...regulation of sporulation / dinitrosyl-iron complex binding / bacterial-type RNA polymerase core enzyme binding / sigma factor activity / 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 / cell cycle / cell division / DNA-templated transcription / regulation of DNA-templated transcription / positive regulation of DNA-templated transcription / magnesium ion binding / DNA binding / zinc ion binding / metal ion binding / cytoplasm
Similarity search - Function
Sporulation regulator WhiA / Sporulation transcription regulator WhiA, N-terminal domain / Sporulation regulator WhiA, C-terminal / WhiA, LAGLIDADG-like domain / WhiA C-terminal HTH domain / WhiA N-terminal LAGLIDADG-like domain / WhiA LAGLIDADG-like domain / Transcription factor WhiB / WhiB-like iron-sulfur binding domain / Transcription factor WhiB ...Sporulation regulator WhiA / Sporulation transcription regulator WhiA, N-terminal domain / Sporulation regulator WhiA, C-terminal / WhiA, LAGLIDADG-like domain / WhiA C-terminal HTH domain / WhiA N-terminal LAGLIDADG-like domain / WhiA LAGLIDADG-like domain / Transcription factor WhiB / WhiB-like iron-sulfur binding domain / Transcription factor WhiB / 4Fe-4S WhiB-like (Wbl)-type iron-sulfur binding domain profile. / RNA polymerase-binding protein RbpA / RbpA superfamily / RNA polymerase-binding protein / Homing endonuclease / 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) / Probable cell division protein WhiA / DNA-directed RNA polymerase subunit beta' / Transcriptional regulator WhiB / RNA polymerase sigma factor SigA / RNA polymerase-binding protein RbpA / DNA-directed RNA polymerase subunit omega / DNA-directed RNA polymerase subunit beta / DNA-directed RNA polymerase subunit alpha
Similarity search - Component
Biological speciesStreptomyces venezuelae ATCC 10712 (bacteria)
Streptomyces venezuelae (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.12 Å
AuthorsLilic, M. / 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: Proc Natl Acad Sci U S A / Year: 2023
Title: Structural basis of dual activation of cell division by the actinobacterial transcription factors WhiA and WhiB.
Authors: Mirjana Lilic / Neil A Holmes / Matthew J Bush / Alexandra K Marti / David A Widdick / Kim C Findlay / Young Joo Choi / Ruby Froom / Steven Koh / Mark J Buttner / Elizabeth A Campbell /
Abstract: Studies of transcriptional initiation in different bacterial clades reveal diverse molecular mechanisms regulating this first step in gene expression. The WhiA and WhiB factors are both required to ...Studies of transcriptional initiation in different bacterial clades reveal diverse molecular mechanisms regulating this first step in gene expression. The WhiA and WhiB factors are both required to express cell division genes in Actinobacteria and are essential in notable pathogens such as . The WhiA/B regulons and binding sites have been elucidated in (), where they coordinate to activate sporulation septation. However, how these factors cooperate at the molecular level is not understood. Here we present cryoelectron microscopy structures of transcriptional regulatory complexes comprising RNA polymerase (RNAP) σ-holoenzyme and WhiA and WhiB, in complex with the WhiA/B target promoter . These structures reveal that WhiB binds to domain 4 of σ (σ) of the σ-holoenzyme, bridging an interaction with WhiA while making non-specific contacts with the DNA upstream of the -35 core promoter element. The N-terminal homing endonuclease-like domain of WhiA interacts with WhiB, while the WhiA C-terminal domain (WhiA-CTD) makes base-specific contacts with the conserved WhiA GACAC motif. Notably, the structure of the WhiA-CTD and its interactions with the WhiA motif are strikingly similar to those observed between σ housekeeping σ-factors and the -35 promoter element, suggesting an evolutionary relationship. Structure-guided mutagenesis designed to disrupt these protein-DNA interactions reduces or abolishes developmental cell division in confirming their significance. Finally, we compare the architecture of the WhiA/B σ-holoenzyme promoter complex with the unrelated but model CAP Class I and Class II complexes, showing that WhiA/WhiB represent a new mechanism in bacterial transcriptional activation.
History
DepositionAug 3, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 1, 2023Provider: repository / Type: Initial release
Revision 1.1Mar 22, 2023Group: 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

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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: RNA polymerase sigma factor SigA
G: RNA polymerase-binding protein RbpA
H: Transcriptional regulator WhiB
I: Probable cell division protein WhiA
O: DNA (38-MER)
P: DNA (38-MER)
Q: DNA (38-MER)
R: DNA (38-MER)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)535,00918
Polymers534,43713
Non-polymers5725
Water0
1


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

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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: 36734.641 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Streptomyces venezuelae ATCC 10712 (bacteria)
Strain: ATCC 10712 / CBS 650.69 / DSM 40230 / JCM 4526 / NBRC 13096 / PD 04745
Gene: rpoA, SVEN_4407 / Production host: Escherichia coli (E. coli) / References: UniProt: F2RJV9, DNA-directed RNA polymerase
#2: Protein DNA-directed RNA polymerase subunit beta / Polymerase / RNAP subunit beta / RNA polymerase subunit beta / Transcriptase subunit beta


Mass: 130458.953 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Streptomyces venezuelae (bacteria)
Strain: ATCC 10712 / CBS 650.69 / DSM 40230 / JCM 4526 / NBRC 13096 / PD 04745
References: UniProt: F2RIS5, DNA-directed RNA polymerase
#3: Protein DNA-directed RNA polymerase subunit beta' / Polymerase / RNAP subunit beta' / RNA polymerase subunit beta' / Transcriptase subunit beta'


Mass: 144614.781 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Streptomyces venezuelae (bacteria)
References: UniProt: A0A5P2AAC9, DNA-directed RNA polymerase
#4: Protein DNA-directed RNA polymerase subunit omega / Polymerase / RNAP omega subunit / RNA polymerase omega subunit / Transcriptase subunit omega


Mass: 9730.968 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Streptomyces venezuelae (bacteria)
Strain: ATCC 10712 / CBS 650.69 / DSM 40230 / JCM 4526 / NBRC 13096 / PD 04745
References: UniProt: F2RCK7, DNA-directed RNA polymerase

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Protein , 4 types, 4 molecules FGHI

#5: Protein RNA polymerase sigma factor SigA


Mass: 56696.156 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Streptomyces venezuelae (bacteria)
Strain: ATCC 10712 / CBS 650.69 / DSM 40230 / JCM 4526 / NBRC 13096 / PD 04745
Gene: sigA, SVEN_5499 / Production host: Escherichia coli (E. coli) / References: UniProt: F2R7X6
#6: Protein RNA polymerase-binding protein RbpA


Mass: 14116.112 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Streptomyces venezuelae (bacteria)
Strain: ATCC 10712 / CBS 650.69 / DSM 40230 / JCM 4526 / NBRC 13096 / PD 04745
Gene: rbpA, SVEN_1012 / Production host: Escherichia coli (E. coli) / References: UniProt: F2RBH1
#7: Protein Transcriptional regulator WhiB


Mass: 10011.238 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Streptomyces venezuelae (bacteria) / Gene: whiB, DEJ44_12835, DEJ45_22255, DEJ46_24450 / Production host: Escherichia coli (E. coli) / References: UniProt: A0A5P2AC98
#8: Protein Probable cell division protein WhiA


Mass: 34959.152 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Streptomyces venezuelae (bacteria) / Gene: whiA, DEJ44_07690, DEJ45_27525 / Production host: Escherichia coli (E. coli) / References: UniProt: A0A5P1ZKC3

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DNA chain , 2 types, 4 molecules OQPR

#9: DNA chain DNA (38-MER)


Mass: 15068.661 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) Streptomyces venezuelae (bacteria)
#10: DNA chain DNA (38-MER)


Mass: 15121.666 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) Streptomyces venezuelae (bacteria)

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Non-polymers , 3 types, 5 molecules

#11: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 3 / 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

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Details

Has ligand of interestN

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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: Streptomyces venezuelae RNAP unconstrained open promoter complex with WhiA and WhiB transcription factors
Type: COMPLEX / Entity ID: #1-#10 / Source: NATURAL
Source (natural)Organism: Streptomyces venezuelae (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 FIELDBright-field microscopy / Nominal defocus max: 2500 nm / Nominal defocus min: 500 nm
Image recordingElectron dose: 66 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.19.2_4158: / Classification: refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.12 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 118181 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00333116
ELECTRON MICROSCOPYf_angle_d0.48845396
ELECTRON MICROSCOPYf_dihedral_angle_d17.4325562
ELECTRON MICROSCOPYf_chiral_restr0.045162
ELECTRON MICROSCOPYf_plane_restr0.0035461

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