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- PDB-7nbn: Allostery through DNA drives phenotype switching -

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Basic information

Entry
Database: PDB / ID: 7nbn
TitleAllostery through DNA drives phenotype switching
Components(AddAB promoter) x 2
KeywordsDNA BINDING PROTEIN / Transcription-factor / DNA-binding / A-tract / Allostery
Function / homologyDNA / DNA (> 10)
Function and homology information
Biological speciesBacillus subtilis (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 7 Å
AuthorsRosenblum, G. / Elad, N. / Rozenberg, H. / Wiggers, F. / Jungwirth, J. / Hofmann, H.
Funding support Israel, 1items
OrganizationGrant numberCountry
Israel Science Foundation1549/15 Israel
CitationJournal: Nat Commun / Year: 2021
Title: Allostery through DNA drives phenotype switching.
Authors: Gabriel Rosenblum / Nadav Elad / Haim Rozenberg / Felix Wiggers / Jakub Jungwirth / Hagen Hofmann /
Abstract: Allostery is a pervasive principle to regulate protein function. Growing evidence suggests that also DNA is capable of transmitting allosteric signals. Yet, whether and how DNA-mediated allostery ...Allostery is a pervasive principle to regulate protein function. Growing evidence suggests that also DNA is capable of transmitting allosteric signals. Yet, whether and how DNA-mediated allostery plays a regulatory role in gene expression remained unclear. Here, we show that DNA indeed transmits allosteric signals over long distances to boost the binding cooperativity of transcription factors. Phenotype switching in Bacillus subtilis requires an all-or-none promoter binding of multiple ComK proteins. We use single-molecule FRET to demonstrate that ComK-binding at one promoter site increases affinity at a distant site. Cryo-EM structures of the complex between ComK and its promoter demonstrate that this coupling is due to mechanical forces that alter DNA curvature. Modifications of the spacer between sites tune cooperativity and show how to control allostery, which allows a fine-tuning of the dynamic properties of genetic circuits.
History
DepositionJan 27, 2021Deposition site: PDBE / Processing site: PDBE
Revision 1.0Apr 7, 2021Provider: repository / Type: Initial release
Revision 1.1Jun 2, 2021Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _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

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Structure viewerMolecule:
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Assembly

Deposited unit
A: AddAB promoter
B: AddAB promoter


Theoretical massNumber of molelcules
Total (without water)48,0972
Polymers48,0972
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: fluorescence resonance energy transfer, native gel electrophoresis
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area7080 Å2
ΔGint-38 kcal/mol
Surface area27740 Å2
MethodPISA

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Components

#1: DNA chain AddAB promoter


Mass: 24230.533 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Bacillus subtilis (bacteria)
#2: DNA chain AddAB promoter


Mass: 23866.352 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Bacillus subtilis (bacteria)

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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: ComK transcription factor bound to its comG promoter DNA.
Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Bacillus subtilis (bacteria)
Source (recombinant)Organism: Synthetic construct (others)
Buffer solutionpH: 7
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMTrisC4H11NO31
250 mMArginineC6H14N4O21
3150 mMKClKCl1
45 mMMgCl2MgCl21
53 mMDithiothreitolC4H10O2S21
60.001 %Tween-20C58H114O261
SpecimenConc.: 0.24 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: Monodisperse complex with dsDNA and 4 ComK equivalents.
Specimen supportGrid type: UltrAuFoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 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 FIELDBright-field microscopy / Nominal magnification: 105000 X / Calibrated magnification: 58207 X / Cs: 2.7 mm / C2 aperture diameter: 70 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 2 sec. / Electron dose: 55.7 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.19_4092: / Classification: refinement
EM software
IDNameVersionCategory
2SerialEM3.8image acquisition
4cryoSPARCCTF correction
7Coot0.9.2model fitting
9Coot0.9.2model refinement
10cryoSPARCinitial Euler assignment
11cryoSPARCfinal Euler assignment
13cryoSPARC3D reconstruction
14PHENIX1.19model fitting
15PHENIX1.19model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 130000
3D reconstructionResolution: 7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 100695 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0043586
ELECTRON MICROSCOPYf_angle_d0.575530
ELECTRON MICROSCOPYf_dihedral_angle_d34.4541554
ELECTRON MICROSCOPYf_chiral_restr0.026624
ELECTRON MICROSCOPYf_plane_restr0.002156

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