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- PDB-7ckq: The cryo-EM structure of B. subtilis BmrR transcription activatio... -

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Basic information

Entry
Database: PDB / ID: 7ckq
TitleThe cryo-EM structure of B. subtilis BmrR transcription activation complex
Components
  • (DNA (50-MER)) x 2
  • (DNA-directed RNA polymerase subunit ...) x 4
  • Multidrug-efflux transporter 1 regulator
  • RNA polymerase sigma factor SigA
  • UPF0356 protein YkzG
KeywordsTRANSCRIPTION / RNA polymerase / Transcription activation
Function / homology
Function and homology information


nucleoid / sigma factor activity / bacterial-type RNA polymerase core enzyme binding / cytosolic DNA-directed RNA polymerase complex / DNA-directed RNA polymerase complex / DNA-templated transcription initiation / ribonucleoside binding / DNA-directed RNA polymerase / DNA-directed RNA polymerase activity / protein dimerization activity ...nucleoid / sigma factor activity / bacterial-type RNA polymerase core enzyme binding / cytosolic DNA-directed RNA polymerase complex / DNA-directed RNA polymerase complex / DNA-templated transcription initiation / ribonucleoside binding / DNA-directed RNA polymerase / DNA-directed RNA polymerase activity / protein dimerization activity / DNA-binding transcription factor activity / response to antibiotic / DNA-templated transcription / regulation of DNA-templated transcription / magnesium ion binding / DNA binding / zinc ion binding / cytoplasm
Similarity search - Function
RNA polymerase epsilon subunit / RNA polymerase epsilon subunit / GyrI-like small molecule binding domain / GyrI-like small molecule binding domain / MerR family regulatory protein / Regulatory factor, effector binding domain superfamily / MerR-type HTH domain signature. / : / MerR HTH family regulatory protein / helix_turn_helix, mercury resistance ...RNA polymerase epsilon subunit / RNA polymerase epsilon subunit / GyrI-like small molecule binding domain / GyrI-like small molecule binding domain / MerR family regulatory protein / Regulatory factor, effector binding domain superfamily / MerR-type HTH domain signature. / : / MerR HTH family regulatory protein / helix_turn_helix, mercury resistance / MerR-type HTH domain profile. / MerR-type HTH domain / RNA polymerase sigma factor 70, region 1.1 / Sigma-70 factor, region 1.1 superfamily / Sigma-70 factor, region 1.1 / Putative DNA-binding domain superfamily / 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 / 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, 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 / DNA-directed RNA polymerase, insert domain superfamily / RNA polymerase Rpb1, domain 2 / RNA polymerase Rpb1, domain 5 / RNA polymerase Rpb1, domain 4 / RNA polymerase Rpb2, domain 2 / RNA polymerase Rpb2, domain 2 / RNA polymerase, RBP11-like subunit / RNA polymerase, N-terminal / RNA polymerase Rpb1, funnel domain superfamily / RNA polymerase I subunit A N-terminus / 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
TETRAPHENYLPHOSPHONIUM / DNA / DNA (> 10) / DNA-directed RNA polymerase subunit epsilon / DNA-directed RNA polymerase subunit omega / RNA polymerase sigma factor SigA / DNA-directed RNA polymerase subunit alpha / DNA-directed RNA polymerase subunit beta / DNA-directed RNA polymerase subunit beta' / Multidrug-efflux transporter 1 regulator
Similarity search - Component
Biological speciesBacillus subtilis (bacteria)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.4 Å
AuthorsFang, C.L. / Zhang, Y.
Funding support China, 1items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC)31822001 China
CitationJournal: Nat Commun / Year: 2020
Title: The bacterial multidrug resistance regulator BmrR distorts promoter DNA to activate transcription.
Authors: Chengli Fang / Linyu Li / Yihan Zhao / Xiaoxian Wu / Steven J Philips / Linlin You / Mingkang Zhong / Xiaojin Shi / Thomas V O'Halloran / Qunyi Li / Yu Zhang /
Abstract: The MerR-family proteins represent a unique family of bacteria transcription factors (TFs), which activate transcription in a manner distinct from canonical ones. Here, we report a cryo-EM structure ...The MerR-family proteins represent a unique family of bacteria transcription factors (TFs), which activate transcription in a manner distinct from canonical ones. Here, we report a cryo-EM structure of a B. subtilis transcription activation complex comprising B. subtilis six-subunit (2αββ'ωε) RNA Polymerase (RNAP) core enzyme, σ, a promoter DNA, and the ligand-bound B. subtilis BmrR, a prototype of MerR-family TFs. The structure reveals that RNAP and BmrR recognize the upstream promoter DNA from opposite faces and induce four significant kinks from the -35 element to the -10 element of the promoter DNA in a cooperative manner, which restores otherwise inactive promoter activity by shortening the length of promoter non-optimal -35/-10 spacer. Our structure supports a DNA-distortion and RNAP-non-contact paradigm of transcriptional activation by MerR TFs.
History
DepositionJul 18, 2020Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Dec 16, 2020Provider: repository / Type: Initial release
Revision 1.1Dec 23, 2020Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation_author.name
Revision 1.2Mar 27, 2024Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_3d_fitting_list / pdbx_initial_refinement_model
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type

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Structure visualization

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  • Deposited structure unit
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  • Superimposition on EM map
  • EMDB-30390
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Structure viewerMolecule:
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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
1: DNA (50-MER)
2: DNA (50-MER)
G: Multidrug-efflux transporter 1 regulator
I: Multidrug-efflux transporter 1 regulator
H: UPF0356 protein YkzG
hetero molecules


Theoretical massNumber of molelcules
Total (without water)494,72816
Polymers493,89511
Non-polymers8345
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area63370 Å2
ΔGint-393 kcal/mol
Surface area159080 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 / RNAP subunit alpha / RNA polymerase subunit alpha / Transcriptase subunit alpha


Mass: 34842.387 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Bacillus subtilis (strain 168) (bacteria) / Strain: 168 / References: UniProt: P20429, DNA-directed RNA polymerase
#2: Protein DNA-directed RNA polymerase subunit beta / RNAP subunit beta / RNA polymerase subunit beta / Transcriptase subunit beta


Mass: 133847.938 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Bacillus subtilis (strain 168) (bacteria) / Strain: 168 / References: UniProt: P37870, DNA-directed RNA polymerase
#3: Protein DNA-directed RNA polymerase subunit beta' / RNAP subunit beta' / RNA polymerase subunit beta' / Transcriptase subunit beta'


Mass: 134444.953 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Bacillus subtilis (strain 168) (bacteria) / Strain: 168 / References: UniProt: P37871, DNA-directed RNA polymerase
#4: Protein DNA-directed RNA polymerase subunit omega / RNAP omega subunit / RNA polymerase omega subunit / Transcriptase subunit omega


Mass: 7766.921 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Bacillus subtilis (strain 168) (bacteria) / Strain: 168 / References: UniProt: O35011, DNA-directed RNA polymerase

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Protein , 3 types, 4 molecules FGIH

#5: Protein RNA polymerase sigma factor SigA / Sigma-43 / Sigma-A


Mass: 43007.406 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Bacillus subtilis (strain 168) (bacteria) / Strain: 168 / References: UniProt: P06224
#8: Protein Multidrug-efflux transporter 1 regulator


Mass: 32951.688 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Bacillus subtilis (strain 168) (bacteria)
Strain: 168 / Gene: bmrR, bmr1R, BSU24020 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P39075
#9: Protein UPF0356 protein YkzG


Mass: 8263.358 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Bacillus subtilis (strain 168) (bacteria) / Strain: 168 / References: UniProt: O31718

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DNA chain , 2 types, 2 molecules 12

#6: DNA chain DNA (50-MER)


Mass: 15489.907 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#7: DNA chain DNA (50-MER)


Mass: 15485.940 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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Non-polymers , 3 types, 5 molecules

#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
#12: Chemical ChemComp-P4P / TETRAPHENYLPHOSPHONIUM


Mass: 339.389 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C24H20P

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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: Bacillus subtilis BmrR transcription activation complex
Type: COMPLEX / Entity ID: #1-#9 / Source: RECOMBINANT
Molecular weightValue: 0.48 MDa / Experimental value: NO
Source (natural)Organism: Bacillus subtilis subsp. subtilis str. 168 (bacteria)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
110 mMHEPESC8H18N2O4S1
250 mMpotassium chlorideKCl1
35 mMmagnesium chlorideMgCl21
43 mMDL-DithiothreitolC4H10O2S21
SpecimenConc.: 19 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: C-flat-1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 282.65 K

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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 / Cs: 2.7 mm
Image recordingAverage exposure time: 7.6 sec. / Electron dose: 60.8 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 2226
Image scansMovie frames/image: 38

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Processing

EM software
IDNameVersionCategory
2SerialEMimage acquisition
4CTFFIND4CTF correction
7UCSF Chimeramodel fitting
9RELION3initial Euler assignment
10RELION3final Euler assignment
11RELION3classification
12RELION33D reconstruction
13PHENIXmodel refinement
CTF correctionType: NONE
Particle selectionNum. of particles selected: 494295
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 4.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 103226 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT
Atomic model building
IDPDB-ID 3D fitting-IDAccession codeInitial refinement model-IDSource nameType
16LDI

6ldi

16LDI1PDBexperimental model
21EXI

1exi

11EXI2PDBexperimental model
34NJC

4njc

14NJC3PDBexperimental model

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