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- PDB-6wmr: F. tularensis RNAPs70-(MglA-SspA)-iglA DNA complex -

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Basic information

Entry
Database: PDB / ID: 6wmr
TitleF. tularensis RNAPs70-(MglA-SspA)-iglA DNA complex
Components
  • (DNA-directed RNA polymerase subunit ...) x 5
  • DNA NT-strand
  • DNA NT-strand downstream
  • DNA T-strand
  • DNA T-strand downstream
  • Macrophage growth locus, subunit A
  • RNA polymerase sigma factor RpoD
  • Stringent starvation protein A
KeywordsTRANSCRIPTION / RNA polymerase complex
Function / homology
Function and homology information


sigma factor activity / glutathione metabolic process / DNA-directed RNA polymerase complex / DNA-templated transcription initiation / ribonucleoside binding / DNA-directed RNA polymerase / DNA-directed RNA polymerase activity / transferase activity / protein dimerization activity / DNA-templated transcription ...sigma factor activity / glutathione metabolic process / DNA-directed RNA polymerase complex / DNA-templated transcription initiation / ribonucleoside binding / DNA-directed RNA polymerase / DNA-directed RNA polymerase activity / transferase activity / protein dimerization activity / DNA-templated transcription / magnesium ion binding / DNA binding / zinc ion binding / cytoplasm
Similarity search - Function
: / Macrophage Locus Protein A, C-terminal domain / Glutathione S-transferase, N-terminal domain / Glutathione S-transferase, N-terminal domain / RNA polymerase sigma factor 70, non-essential domain / Sigma-70, non-essential region / RNA polymerase sigma factor 70, region 1.1 / Sigma-70 factor, region 1.1 superfamily / Sigma-70 factor, region 1.1 / RNA polymerase Rpb1 funnel domain ...: / Macrophage Locus Protein A, C-terminal domain / Glutathione S-transferase, N-terminal domain / Glutathione S-transferase, N-terminal domain / RNA polymerase sigma factor 70, non-essential domain / Sigma-70, non-essential region / RNA polymerase sigma factor 70, region 1.1 / Sigma-70 factor, region 1.1 superfamily / Sigma-70 factor, region 1.1 / RNA polymerase Rpb1 funnel domain / Glutathione S-transferase, C-terminal domain / Sigma-70 factors family signature 1. / Topoisomerase I; Chain A, domain 4 / 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 / Glutathione transferase family / 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 / Glutathione S-transferase Yfyf (Class Pi); Chain A, domain 2 - #10 / RNA polymerase sigma factor, region 3/4-like / Glutathione S-transferase, C-terminal / Glutathione S-transferase Yfyf (Class Pi); Chain A, domain 2 / Glutathione S-transferase, C-terminal-like / Soluble glutathione S-transferase C-terminal domain profile. / Soluble glutathione S-transferase N-terminal domain profile. / Glutathione S-transferase, N-terminal / Glutathione S-transferase, C-terminal domain 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 beta subunit, bacterial-type / DNA-directed RNA polymerase, alpha subunit / Glutaredoxin / 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 / Glutaredoxin / 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, beta subunit, protrusion / RNA polymerase beta subunit / RNA polymerase Rpb1, domain 1 / 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 Rpb2, domain 2 / RNA polymerase Rpb2, domain 2 / RNA polymerase, N-terminal / RNA polymerase Rpb1, funnel domain superfamily / 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 / Thioredoxin-like superfamily / Winged helix-like DNA-binding domain superfamily / Up-down Bundle / Orthogonal Bundle / 3-Layer(aba) Sandwich / Mainly Alpha / Alpha Beta
Similarity search - Domain/homology
DNA / DNA (> 10) / RNA polymerase sigma factor RpoD / GST N-terminal domain-containing protein / Glutathione S-transferase / DNA-directed RNA polymerase subunit beta / DNA-directed RNA polymerase subunit beta' / DNA-directed RNA polymerase subunit omega / DNA-directed RNA polymerase subunit alpha 2 / DNA-directed RNA polymerase subunit alpha 1
Similarity search - Component
Biological speciesFrancisella tularensis subsp. holarctica LVS (bacteria)
Francisella tularensis subsp. holarctica (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.46 Å
AuthorsTravis, B.A. / Brennan, R.G. / Schumacher, M.A.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM130290 United States
CitationJournal: Mol Cell / Year: 2021
Title: Structural Basis for Virulence Activation of Francisella tularensis.
Authors: Brady A Travis / Kathryn M Ramsey / Samantha M Prezioso / Thomas Tallo / Jamie M Wandzilak / Allen Hsu / Mario Borgnia / Alberto Bartesaghi / Simon L Dove / Richard G Brennan / Maria A Schumacher /
Abstract: The bacterium Francisella tularensis (Ft) is one of the most infectious agents known. Ft virulence is controlled by a unique combination of transcription regulators: the MglA-SspA heterodimer, PigR, ...The bacterium Francisella tularensis (Ft) is one of the most infectious agents known. Ft virulence is controlled by a unique combination of transcription regulators: the MglA-SspA heterodimer, PigR, and the stress signal, ppGpp. MglA-SspA assembles with the σ-associated RNAP holoenzyme (RNAPσ), forming a virulence-specialized polymerase. These factors activate Francisella pathogenicity island (FPI) gene expression, which is required for virulence, but the mechanism is unknown. Here we report FtRNAPσ-promoter-DNA, FtRNAPσ-(MglA-SspA)-promoter DNA, and FtRNAPσ-(MglA-SspA)-ppGpp-PigR-promoter DNA cryo-EM structures. Structural and genetic analyses show MglA-SspA facilitates σ binding to DNA to regulate virulence and virulence-enhancing genes. Our Escherichia coli RNAPσhomodimeric EcSspA structure suggests this is a general SspA-transcription regulation mechanism. Strikingly, our FtRNAPσ-(MglA-SspA)-ppGpp-PigR-DNA structure reveals ppGpp binding to MglA-SspA tethers PigR to promoters. PigR in turn recruits FtRNAP αCTDs to DNA UP elements. Thus, these studies unveil a unique mechanism for Ft pathogenesis involving a virulence-specialized RNAP that employs two (MglA-SspA)-based strategies to activate virulence genes.
History
DepositionApr 21, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Nov 11, 2020Provider: repository / Type: Initial release
Revision 1.1Dec 2, 2020Group: Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.2Jan 20, 2021Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.year
Revision 1.3Mar 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

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Structure visualization

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  • Deposited structure unit
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Structure viewerMolecule:
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Assembly

Deposited unit
G: DNA NT-strand
H: DNA T-strand
X: DNA NT-strand downstream
J: DNA T-strand downstream
S: Stringent starvation protein A
M: Macrophage growth locus, subunit A
Z: RNA polymerase sigma factor RpoD
A: DNA-directed RNA polymerase subunit alpha 1
B: DNA-directed RNA polymerase subunit alpha 2
C: DNA-directed RNA polymerase subunit beta
D: DNA-directed RNA polymerase subunit beta'
E: DNA-directed RNA polymerase subunit omega
hetero molecules


Theoretical massNumber of molelcules
Total (without water)528,29215
Polymers528,13712
Non-polymers1553
Water00
1


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

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Components

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DNA chain , 4 types, 4 molecules GHXJ

#1: DNA chain DNA NT-strand


Mass: 10819.960 Da / Num. of mol.: 1 / Source method: obtained synthetically
Source: (synth.) Francisella tularensis subsp. holarctica LVS (bacteria)
#2: DNA chain DNA T-strand


Mass: 7283.774 Da / Num. of mol.: 1 / Source method: obtained synthetically
Source: (synth.) Francisella tularensis subsp. holarctica LVS (bacteria)
#3: DNA chain DNA NT-strand downstream


Mass: 3350.185 Da / Num. of mol.: 1 / Source method: obtained synthetically
Source: (synth.) Francisella tularensis subsp. holarctica LVS (bacteria)
#4: DNA chain DNA T-strand downstream


Mass: 3359.199 Da / Num. of mol.: 1 / Source method: obtained synthetically
Source: (synth.) Francisella tularensis subsp. holarctica LVS (bacteria)

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Protein , 3 types, 3 molecules SMZ

#5: Protein Stringent starvation protein A


Mass: 24098.213 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Source: (natural) Francisella tularensis subsp. holarctica LVS (bacteria)
References: UniProt: A0A3N3ZVL6
#6: Protein Macrophage growth locus, subunit A


Mass: 23650.656 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Source: (natural) Francisella tularensis subsp. holarctica LVS (bacteria)
References: UniProt: A0A0B6CT24
#7: Protein RNA polymerase sigma factor RpoD / Sigma-70


Mass: 67748.469 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Source: (natural) Francisella tularensis subsp. holarctica LVS (bacteria)
References: UniProt: A0A0B3WH85

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DNA-directed RNA polymerase subunit ... , 5 types, 5 molecules ABCDE

#8: Protein DNA-directed RNA polymerase subunit alpha 1 / RNAP subunit alpha 1 / RNA polymerase subunit alpha 1 / Transcriptase subunit alpha 1


Mass: 35393.387 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Source: (natural) Francisella tularensis subsp. holarctica (strain LVS) (bacteria)
Strain: LVS / References: UniProt: Q2A5E5, DNA-directed RNA polymerase
#9: Protein DNA-directed RNA polymerase subunit alpha 2 / RNAP subunit alpha 2 / RNA polymerase subunit alpha 2 / Transcriptase subunit alpha 2


Mass: 35113.031 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Source: (natural) Francisella tularensis subsp. holarctica (strain LVS) (bacteria)
Strain: LVS / References: UniProt: Q2A4H7, DNA-directed RNA polymerase
#10: Protein DNA-directed RNA polymerase subunit beta / RNAP subunit beta / RNA polymerase subunit beta / Transcriptase subunit beta


Mass: 151536.141 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Source: (natural) Francisella tularensis subsp. holarctica (strain LVS) (bacteria)
Strain: LVS / References: UniProt: Q2A1M7, DNA-directed RNA polymerase
#11: Protein DNA-directed RNA polymerase subunit beta' / RNAP subunit beta' / RNA polymerase subunit beta' / Transcriptase subunit beta'


Mass: 157599.578 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Source: (natural) Francisella tularensis subsp. holarctica (strain LVS) (bacteria)
Strain: LVS / References: UniProt: Q2A1M8, DNA-directed RNA polymerase
#12: Protein DNA-directed RNA polymerase subunit omega / RNAP omega subunit / RNA polymerase omega subunit / Transcriptase subunit omega


Mass: 8184.333 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Source: (natural) Francisella tularensis subsp. holarctica (strain LVS) (bacteria)
Strain: LVS / References: UniProt: Q2A273, DNA-directed RNA polymerase

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Non-polymers , 2 types, 3 molecules

#13: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg
#14: 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 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: Francisella RNAPs70-(MglA-SspA)-iglA complex / Type: COMPLEX / Entity ID: #1-#12 / Source: NATURAL
Molecular weightExperimental value: NO
Source (natural)Organism: Francisella tularensis subsp. holarctica LVS (bacteria)
Buffer solutionpH: 8
SpecimenConc.: 0.4 mg/ml / Embedding 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: OTHER
Electron lensMode: BRIGHT FIELD / Cs: 2.7 mm
Image recordingElectron dose: 42 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON III (4k x 4k)

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Processing

Software
NameVersionClassification
phenix.real_space_refine1.17rc5_3630refinement
PHENIX1.17rc5_3630refinement
EM software
IDNameVersionCategory
7Coot0.9model fitting
9PHENIX1.17model refinement
10RELION3initial Euler assignment
11cisTEM1.0.0final Euler assignment
13cisTEM1.0.03D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.46 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 21457 / Symmetry type: POINT
Atomic model buildingSpace: REAL
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 128.39 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.008231047
ELECTRON MICROSCOPYf_angle_d0.747142279
ELECTRON MICROSCOPYf_chiral_restr0.04634927
ELECTRON MICROSCOPYf_plane_restr0.00435168
ELECTRON MICROSCOPYf_dihedral_angle_d21.71264868

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