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- PDB-8hvr: Cryo-EM structure of AfsR-dependent transcription activation comp... -

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Basic information

Entry
Database: PDB / ID: 8hvr
TitleCryo-EM structure of AfsR-dependent transcription activation complex with afsS promoter
Components
  • (DNA (65-MER)) x 2
  • (DNA-directed RNA polymerase subunit ...) x 4
  • Putative transcriptional factor regulator
  • RNA polymerase principal sigma factor HrdB
  • RNA polymerase-binding protein RbpA
  • Regulatory protein AfsR
KeywordsGENE REGULATION / RNA polymerase / SARP regulator / TRANSCRIPTION
Function / homology
Function and homology information


pigment binding / bacterial-type RNA polymerase holo enzyme binding / phosphorelay signal transduction system / sigma factor activity / rRNA transcription / bacterial-type RNA polymerase core enzyme binding / cytosolic DNA-directed RNA polymerase complex / antibiotic biosynthetic process / DNA-directed RNA polymerase complex / DNA-templated transcription initiation ...pigment binding / bacterial-type RNA polymerase holo enzyme binding / phosphorelay signal transduction system / sigma factor activity / rRNA transcription / bacterial-type RNA polymerase core enzyme binding / cytosolic DNA-directed RNA polymerase complex / antibiotic biosynthetic process / DNA-directed RNA polymerase complex / DNA-templated transcription initiation / ADP binding / ribonucleoside binding / DNA-directed RNA polymerase / DNA-directed RNA polymerase activity / protein dimerization activity / DNA-templated transcription / regulation of DNA-templated transcription / magnesium ion binding / DNA binding / zinc ion binding / ATP binding / cytoplasm
Similarity search - Function
Bacterial transcriptional activator domain / : / Bacterial transcriptional activator domain / Bacterial transcriptional activator domain / CarD-like, C-terminal domain / : / : / CarD, C-terminal domain / CarD-like/TRCF, RNAP-interacting domain / CarD-like/TRCF, RNAP-interacting domain superfamily ...Bacterial transcriptional activator domain / : / Bacterial transcriptional activator domain / Bacterial transcriptional activator domain / CarD-like, C-terminal domain / : / : / CarD, C-terminal domain / CarD-like/TRCF, RNAP-interacting domain / CarD-like/TRCF, RNAP-interacting domain superfamily / CarD-like/TRCF RID domain / CarD-like/TRCF domain / Tetratricopeptide repeat / OmpR/PhoB-type DNA-binding domain / NB-ARC / Transcriptional regulatory protein, C terminal / NB-ARC domain / OmpR/PhoB-type DNA-binding domain profile. / Transcriptional regulatory protein, C terminal / Tetratricopeptide repeat / Signal transduction response regulator, C-terminal effector / 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 / TPR repeat region circular profile. / 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 / TPR repeat profile. / Tetratricopeptide repeats / Tetratricopeptide repeat / 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 Rpb1, domain 1 / RNA polymerase, beta subunit, protrusion / RNA polymerase beta subunit / 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, N-terminal / RNA polymerase Rpb2, domain 2 / RNA polymerase Rpb1, funnel domain superfamily / RNA polymerase Rpb2, domain 2 / 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 / Tetratricopeptide-like helical domain superfamily / Winged helix-like DNA-binding domain superfamily / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
DNA / DNA (> 10) / DNA-directed RNA polymerase subunit alpha / : / RNA polymerase principal sigma factor HrdB / Regulatory protein AfsR / DNA-directed RNA polymerase subunit beta' / DNA-directed RNA polymerase subunit omega / DNA-directed RNA polymerase subunit beta / RNA polymerase-binding transcription factor CarD
Similarity search - Component
Biological speciesStreptomyces coelicolor A3
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.35 Å
AuthorsWang, Y. / Zheng, J.
Funding support China, 1items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC)31770068, 32070040 China
CitationJournal: PLoS Biol / Year: 2024
Title: Structural and functional characterization of AfsR, an SARP family transcriptional activator of antibiotic biosynthesis in Streptomyces.
Authors: Yiqun Wang / Xu Yang / Feng Yu / Zixin Deng / Shuangjun Lin / Jianting Zheng /
Abstract: Streptomyces antibiotic regulatory proteins (SARPs) are widely distributed activators of antibiotic biosynthesis. Streptomyces coelicolor AfsR is an SARP regulator with an additional nucleotide- ...Streptomyces antibiotic regulatory proteins (SARPs) are widely distributed activators of antibiotic biosynthesis. Streptomyces coelicolor AfsR is an SARP regulator with an additional nucleotide-binding oligomerization domain (NOD) and a tetratricopeptide repeat (TPR) domain. Here, we present cryo-electron microscopy (cryo-EM) structures and in vitro assays to demonstrate how the SARP domain activates transcription and how it is modulated by NOD and TPR domains. The structures of transcription initiation complexes (TICs) show that the SARP domain forms a side-by-side dimer to simultaneously engage the afs box overlapping the -35 element and the σHrdB region 4 (R4), resembling a sigma adaptation mechanism. The SARP extensively interacts with the subunits of the RNA polymerase (RNAP) core enzyme including the β-flap tip helix (FTH), the β' zinc-binding domain (ZBD), and the highly flexible C-terminal domain of the α subunit (αCTD). Transcription assays of full-length AfsR and truncated proteins reveal the inhibitory effect of NOD and TPR on SARP transcription activation, which can be eliminated by ATP binding. In vitro phosphorylation hardly affects transcription activation of AfsR, but counteracts the disinhibition of ATP binding. Overall, our results present a detailed molecular view of how AfsR serves to activate transcription.
History
DepositionDec 27, 2022Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Dec 27, 2023Provider: repository / Type: Initial release
Revision 1.1Apr 3, 2024Group: 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

Structure viewerMolecule:
MolmilJmol/JSmol

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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 principal sigma factor HrdB
G: RNA polymerase-binding protein RbpA
H: Putative transcriptional factor regulator
I: Regulatory protein AfsR
J: Regulatory protein AfsR
K: DNA-directed RNA polymerase subunit alpha
O: DNA (65-MER)
P: DNA (65-MER)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)587,73316
Polymers587,57813
Non-polymers1553
Water00
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, 6 molecules ABKCDE

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


Mass: 36734.641 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Streptomyces coelicolor A3(2) (bacteria)
Gene: rpoA, GTW64_13255 / Production host: Escherichia coli BL21(DE3) (bacteria)
References: UniProt: A0A6G2M9E1, DNA-directed RNA polymerase
#2: Protein DNA-directed RNA polymerase subunit beta / RNAP subunit beta / RNA polymerase subunit beta / Transcriptase subunit beta


Mass: 128644.945 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Streptomyces coelicolor A3(2) (bacteria)
Gene: rpoB, SCO4654, SCD82.26 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q9L0L0, DNA-directed RNA polymerase
#3: Protein DNA-directed RNA polymerase subunit beta' / RNAP subunit beta' / RNA polymerase subunit beta' / Transcriptase subunit beta'


Mass: 145912.219 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Streptomyces coelicolor A3(2) (bacteria)
Gene: rpoC, SCO4655, SCD40A.01, SCD82.27 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q8CJT1, DNA-directed RNA polymerase
#4: Protein DNA-directed RNA polymerase subunit omega / RNAP omega subunit / RNA polymerase omega subunit / Transcriptase subunit omega


Mass: 9716.941 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Streptomyces coelicolor A3(2) (bacteria)
Gene: rpoZ, SCO1478, SC9C5.02c / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q9KXS1, DNA-directed RNA polymerase

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Protein , 4 types, 5 molecules FGHIJ

#5: Protein RNA polymerase principal sigma factor HrdB / RNA polymerase sigma factor SigA


Mass: 58188.953 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Streptomyces coelicolor A3(2) (bacteria)
Gene: hrdB, sigA, SCO5820, SC5B8.10 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P18183
#6: Protein RNA polymerase-binding protein RbpA


Mass: 14146.094 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Streptomyces coelicolor A3(2) (bacteria)
Gene: rbpA / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: A0A6M9XI42
#7: Protein Putative transcriptional factor regulator


Mass: 17855.338 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Streptomyces coelicolor A3(2) (bacteria)
Gene: SCD8A.05 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q9L0Q9
#8: Protein Regulatory protein AfsR


Mass: 31479.572 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Streptomyces coelicolor A3(2) (bacteria)
Gene: afsR, afsB, SCO4426, SCD6.04c / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P25941

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DNA chain , 2 types, 2 molecules OP

#9: DNA chain DNA (65-MER)


Mass: 19863.656 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Streptomyces coelicolor A3(2) (bacteria)
#10: DNA chain DNA (65-MER)


Mass: 20086.846 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Streptomyces coelicolor A3(2) (bacteria)

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Non-polymers , 2 types, 3 molecules

#11: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
#12: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Zn / 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: Cryo-EM structure of AfsR-dependent transcription activation complex with afsS promoter
Type: COMPLEX / Entity ID: #1-#5, #7-#10, #6 / Source: RECOMBINANT
Molecular weightValue: 0.56 MDa / Experimental value: YES
Source (natural)Organism: Streptomyces coelicolor A3(2) (bacteria)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMTRISTRIS1
250 mMKClKCl1
33 mMDTTDTT1
45 mMMgCl2MgCl21
SpecimenConc.: 0.8 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 289 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 / Nominal magnification: 130000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm / Alignment procedure: BASIC
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 77 K / Temperature (min): 77 K
Image recordingElectron dose: 40 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 QUANTUM (4k x 4k)
EM imaging opticsEnergyfilter name: GIF Bioquantum
Image scansMovie frames/image: 32

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Processing

SoftwareName: PHENIX / Version: 1.19.2_4158: / Classification: refinement
EM software
IDNameVersionCategoryDetails
2SerialEM3.8.6image acquisition
9PHENIX1.20.1-4487model refinementautomated refinement using real-space maps
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 193644
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.35 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 95223 / Symmetry type: POINT
Atomic model buildingSpace: REAL
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00434829
ELECTRON MICROSCOPYf_angle_d0.68847634
ELECTRON MICROSCOPYf_dihedral_angle_d15.9425671
ELECTRON MICROSCOPYf_chiral_restr0.0455423
ELECTRON MICROSCOPYf_plane_restr0.0055839

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