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- PDB-7vpd: Cryo-EM structure of Streptomyces coelicolor RNAP-promoter open c... -

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Basic information

Entry
Database: PDB / ID: 7vpd
TitleCryo-EM structure of Streptomyces coelicolor RNAP-promoter open complex with one Zur dimers
Components
  • (DNA (84-MER)) x 2
  • (DNA-directed RNA polymerase subunit ...) x 4
  • Putative metal uptake regulation protein
  • RNA polymerase sigma factor SigA
KeywordsDNA BINDING PROTEIN
Function / homology
Function and homology information


regulation of secondary metabolite biosynthetic process / DNA-binding transcription repressor activity / sigma factor activity / DNA-directed RNA polymerase complex / DNA-templated transcription initiation / protein-DNA complex / ribonucleoside binding / DNA-directed 5'-3' RNA polymerase activity / DNA-directed RNA polymerase / transcription cis-regulatory region binding ...regulation of secondary metabolite biosynthetic process / DNA-binding transcription repressor activity / sigma factor activity / DNA-directed RNA polymerase complex / DNA-templated transcription initiation / protein-DNA complex / ribonucleoside binding / DNA-directed 5'-3' RNA polymerase activity / DNA-directed RNA polymerase / transcription cis-regulatory region binding / protein dimerization activity / DNA-binding transcription factor activity / negative regulation of DNA-templated transcription / DNA-templated transcription / magnesium ion binding / DNA binding / zinc ion binding / cytosol / cytoplasm
Similarity search - Function
Ferric-uptake regulator, C-terminal domain / Ferric-uptake regulator / Ferric uptake regulator family / 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 ...Ferric-uptake regulator, C-terminal domain / Ferric-uptake regulator / Ferric uptake regulator family / 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 / RNA polymerase Rpb6 / RNA polymerase, subunit omega/Rpo6/RPB6 / RNA polymerase Rpb6 / RNA polymerase Rpb1, domain 3 superfamily / RPB6/omega subunit-like superfamily / RNA polymerase Rpb1, clamp domain superfamily / RNA polymerase Rpb2, domain 2 superfamily / RNA polymerase Rpb1, domain 3 / RNA polymerase Rpb1, domain 3 / DNA-directed RNA polymerase, subunit beta-prime / RNA polymerase Rpb1, domain 1 / RNA polymerase Rpb1, domain 1 / 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, 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 / RNA polymerase, beta subunit, protrusion / RNA polymerase beta subunit / RNA polymerase, N-terminal / RNA polymerase Rpb1, funnel domain superfamily / RNA polymerase I subunit A N-terminus / DNA-directed RNA polymerase, insert domain superfamily / RNA polymerase, RBP11-like subunit / RNA polymerase Rpb2, domain 2 / RNA polymerase Rpb2, domain 2 / 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 DNA-binding domain superfamily / Winged helix-like DNA-binding domain superfamily
Similarity search - Domain/homology
: / DNA / DNA (> 10) / DNA-directed RNA polymerase subunit alpha / RNA polymerase sigma factor SigA / DNA-directed RNA polymerase subunit beta' / DNA-directed RNA polymerase subunit omega / DNA-directed RNA polymerase subunit beta / Metal uptake regulation protein
Similarity search - Component
Biological speciesStreptomyces coelicolor A3
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.77 Å
AuthorsYang, X. / Zheng, J.
Funding support China, 1items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC)31770068, 32070040 China
CitationJournal: Nucleic Acids Res / Year: 2022
Title: Structural basis of Streptomyces transcription activation by zinc uptake regulator.
Authors: Xu Yang / Yiqun Wang / Guiyang Liu / Zixin Deng / Shuangjun Lin / Jianting Zheng /
Abstract: Streptomyces coelicolor (Sc) is a model organism of actinobacteria to study morphological differentiation and production of bioactive metabolites. Sc zinc uptake regulator (Zur) affects both ...Streptomyces coelicolor (Sc) is a model organism of actinobacteria to study morphological differentiation and production of bioactive metabolites. Sc zinc uptake regulator (Zur) affects both processes by controlling zinc homeostasis. It activates transcription by binding to palindromic Zur-box sequences upstream of -35 elements. Here we deciphered the molecular mechanism by which ScZur interacts with promoter DNA and Sc RNA polymerase (RNAP) by cryo-EM structures and biochemical assays. The ScZur-DNA structures reveal a sequential and cooperative binding of three ScZur dimers surrounding a Zur-box spaced 8 nt upstream from a -35 element. The ScRNAPσHrdB-Zur-DNA structures define protein-protein and protein-DNA interactions involved in the principal housekeeping σHrdB-dependent transcription initiation from a noncanonical promoter with a -10 element lacking the critical adenine residue at position -11 and a TTGCCC -35 element deviating from the canonical TTGACA motif. ScZur interacts with the C-terminal domain of ScRNAP α subunit (αCTD) in a complex structure trapped in an active conformation. Key ScZur-αCTD interfacial residues accounting for ScZur-dependent transcription activation were confirmed by mutational studies. Together, our structural and biochemical results provide a comprehensive model for transcription activation of Zur family regulators.
History
DepositionOct 15, 2021Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Aug 3, 2022Provider: repository / Type: Initial release
Revision 1.1Aug 24, 2022Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID
Revision 1.2Jun 19, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond

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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 sigma factor SigA
M: Putative metal uptake regulation protein
N: Putative metal uptake regulation protein
O: DNA (84-MER)
P: DNA (84-MER)
S: DNA-directed RNA polymerase subunit alpha
hetero molecules


Theoretical massNumber of molelcules
Total (without water)538,94920
Polymers538,40211
Non-polymers5489
Water00
1


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

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Components

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DNA-directed RNA polymerase subunit ... , 4 types, 6 molecules ABSCDE

#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)
Strain: ATCC BAA-471 / A3(2) / M145 / 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)
Strain: ATCC BAA-471 / A3(2) / M145 / 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)
Strain: ATCC BAA-471 / A3(2) / M145 / 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 FMN

#5: Protein RNA polymerase sigma factor SigA


Mass: 58288.078 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Streptomyces coelicolor A3(2) (bacteria)
Gene: sigA, SLI_6088 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: A0A7U9DYK1
#6: Protein Putative metal uptake regulation protein


Mass: 16904.840 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Streptomyces coelicolor A3(2) (bacteria)
Strain: ATCC BAA-471 / A3(2) / M145 / Gene: SCO2508 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q9L2H5

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DNA chain , 2 types, 2 molecules OP

#7: DNA chain DNA (84-MER)


Mass: 25808.424 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Streptomyces coelicolor A3(2) (bacteria) / References: GenBank: 24418971
#8: DNA chain DNA (84-MER)


Mass: 26017.555 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Streptomyces coelicolor A3(2) (bacteria)

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Non-polymers , 2 types, 9 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.: 8 / 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 Streptomyces coelicolor RNAP-promoter open complex with one Zur dimers
Type: COMPLEX / Entity ID: #1-#8 / 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 mMHEPESHEPES1
2100 mMKClKCl1
32 mMDTTDTT1
45 mMMgCl2MgCl21
50.02 mMZnSO4ZnSO41
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
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 defocus max: 2000 nm / Nominal defocus min: 1000 nm
Image recordingElectron dose: 40 e/Å2 / Film or detector model: GATAN K2 QUANTUM (4k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.77 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 19427 / Symmetry type: POINT

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