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- PDB-8k60: Cryo-EM structure of Streptomyces coelicolor transcription initia... -

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Basic information

Entry
Database: PDB / ID: 8k60
TitleCryo-EM structure of Streptomyces coelicolor transcription initiation complex with the global transcription factor AfsR
Components
  • (DNA-directed RNA polymerase subunit ...) x 4
  • Non-template strand DNA for AfsS promoter
  • RNA polymerase principal sigma factor HrdB
  • Regulatory protein AfsR
  • Template strand DNA for AfsS promoter
KeywordsGENE REGULATION / transcription factor / TRANSCRIPTION-DNA complex
Function / homology
Function and homology information


pigment binding / bacterial-type RNA polymerase holo enzyme binding / nucleoid / sigma factor activity / phosphorelay signal transduction system / 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 / nucleoid / sigma factor activity / phosphorelay signal transduction system / 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 / protein dimerization activity / 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 / Tetratricopeptide repeat / NB-ARC / NB-ARC domain / OmpR/PhoB-type DNA-binding domain / Transcriptional regulatory protein, C terminal / OmpR/PhoB-type DNA-binding domain profile. ...Bacterial transcriptional activator domain / : / Bacterial transcriptional activator domain / Bacterial transcriptional activator domain / Tetratricopeptide repeat / NB-ARC / NB-ARC domain / OmpR/PhoB-type DNA-binding domain / Transcriptional regulatory protein, C terminal / 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 / 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 / TPR repeat region circular profile. / 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 Rpb1, funnel domain superfamily / RNA polymerase I subunit A N-terminus / RNA polymerase Rpb2, domain 2 / RNA polymerase Rpb2, domain 2 / 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) / RNA polymerase principal sigma factor HrdB / Regulatory protein AfsR / DNA-directed RNA polymerase subunit alpha / DNA-directed RNA polymerase subunit beta' / DNA-directed RNA polymerase subunit omega / DNA-directed RNA polymerase subunit beta
Similarity search - Component
Biological speciesStreptomyces coelicolor (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsLin, W. / Shi, J.
Funding support China, 1items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC)81903526 China
CitationJournal: iScience / Year: 2024
Title: Structural insights into transcription activation of the antibiotic regulatory protein, AfsR.
Authors: Jing Shi / Zonghang Ye / Zhenzhen Feng / Aijia Wen / Lu Wang / Zhipeng Zhang / Liqiao Xu / Qian Song / Fulin Wang / Tianyu Liu / Shuang Wang / Yu Feng / Wei Lin /
Abstract: The antibiotic regulatory proteins (SARPs) are ubiquitously distributed transcription activators in and control antibiotics biosynthesis and morphological differentiation. However, the molecular ...The antibiotic regulatory proteins (SARPs) are ubiquitously distributed transcription activators in and control antibiotics biosynthesis and morphological differentiation. However, the molecular mechanism behind SARP-dependent transcription initiation remains elusive. We here solve the cryo-EM structure of an AfsR-loading RNA polymerase (RNAP)-promoter intermediate complex (AfsR-RPi) including the RNAP, a large SARP member AfsR, and its target promoter DNA that retains the upstream portion straight. The structure reveals that one dimeric N-terminal AfsR-SARP domain (AfsR-SARP) specifically engages with the same face of the AfsR-binding sites by the conserved DNA-binding domains (DBDs), replacing σR4 to bind the suboptimal -35 element, and shortens the spacer between the -10 and -35 elements. Notably, the AfsR-SARPs also recruit RNAP through extensively interacting with its conserved domains (β flap, σR4, and αCTD). Thus, these macromolecular snapshots support a general model and provide valuable clues for SARP-dependent transcription activation in .
History
DepositionJul 24, 2023Deposition site: PDBJ / Processing site: PDBC
Revision 1.0Jul 31, 2024Provider: repository / Type: Initial release
Revision 1.1Aug 21, 2024Group: Data collection / Database references / Category: citation / em_admin
Item: _citation.page_last / _citation.pdbx_database_id_PubMed ..._citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _em_admin.last_update

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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 principal sigma factor HrdB
G: Non-template strand DNA for AfsS promoter
H: Template strand DNA for AfsS promoter
I: Regulatory protein AfsR
J: Regulatory protein AfsR
K: DNA-directed RNA polymerase subunit alpha
hetero molecules


Theoretical massNumber of molelcules
Total (without water)697,76914
Polymers697,61411
Non-polymers1553
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration, not applicable
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 (strain ATCC BAA-471 / A3(2) / M145) (bacteria)
Gene: rpoA, SCO4729, SC6G4.07 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P60312, 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 (strain ATCC BAA-471 / A3(2) / M145) (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: 144807.078 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Streptomyces coelicolor (strain ATCC BAA-471 / A3(2) / M145) (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 (strain ATCC BAA-471 / A3(2) / M145) (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 , 2 types, 3 molecules FIJ

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


Mass: 56017.578 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Streptomyces coelicolor (strain ATCC BAA-471 / A3(2) / M145) (bacteria)
Gene: hrdB, sigA, SCO5820, SC5B8.10 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P18183
#8: Protein Regulatory protein AfsR


Mass: 105842.578 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Streptomyces coelicolor (strain ATCC BAA-471 / A3(2) / M145) (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 GH

#6: DNA chain Non-template strand DNA for AfsS promoter


Mass: 18177.602 Da / Num. of mol.: 1 / Source method: obtained synthetically
Source: (synth.) Streptomyces coelicolor (strain ATCC BAA-471 / A3(2) / M145) (bacteria)
#7: DNA chain Template strand DNA for AfsS promoter


Mass: 18360.770 Da / Num. of mol.: 1 / Source method: obtained synthetically
Source: (synth.) Streptomyces coelicolor (strain ATCC BAA-471 / A3(2) / M145) (bacteria)

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Non-polymers , 2 types, 3 molecules

#9: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
#10: 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: Streptomyces coelicolor transcription initiation complex with the global transcription factor AfsR
Type: COMPLEX / Entity ID: #1-#8 / Source: MULTIPLE SOURCES
Source (natural)Organism: Streptomyces coelicolor (strain ATCC BAA-471 / A3(2) / M145) (bacteria)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 7.9
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 FIELD / Nominal defocus max: 2000 nm / Nominal defocus min: 1000 nm
Image recordingElectron dose: 52 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

EM softwareName: PHENIX / Version: 1.20.1_4487: / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 231221 / Symmetry type: POINT

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