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- PDB-8x6g: Cryo-EM structure of Staphylococcus aureus sigB-dependent RNAP-pr... -

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Basic information

Entry
Database: PDB / ID: 8x6g
TitleCryo-EM structure of Staphylococcus aureus sigB-dependent RNAP-promoter open complex
Components
  • (DNA (70-mer)) x 2
  • (DNA-directed RNA polymerase subunit ...) x 5
  • Probable DNA-directed RNA polymerase subunit delta
  • RNA polymerase sigma factor
KeywordsTRANSCRIPTION / RNA polymerase / Staphylococcus aureus / sigB
Function / homology
Function and homology information


sigma factor activity / DNA-directed RNA polymerase complex / DNA-templated transcription initiation / 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 ...sigma factor activity / DNA-directed RNA polymerase complex / DNA-templated transcription initiation / 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 / cytoplasm
Similarity search - Function
RNA polymerase sigma-B factor / RNA polymerase sigma-B/F/G type / RNA polymerase epsilon subunit / RNA polymerase epsilon subunit / DNA-directed RNA polymerase subunit delta / DNA-directed RNA polymerase subunit delta, N-terminal domain superfamily / ASXL, HARE-HTH domain / HB1, ASXL, restriction endonuclease HTH domain / HARE-type HTH domain profile. / Sigma-70 factors family signature 1. ...RNA polymerase sigma-B factor / RNA polymerase sigma-B/F/G type / RNA polymerase epsilon subunit / RNA polymerase epsilon subunit / DNA-directed RNA polymerase subunit delta / DNA-directed RNA polymerase subunit delta, N-terminal domain superfamily / ASXL, HARE-HTH domain / HB1, ASXL, restriction endonuclease HTH domain / HARE-type HTH domain profile. / Sigma-70 factors family signature 1. / 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 beta subunit, bacterial-type / DNA-directed RNA polymerase, alpha subunit / 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 / RNA polymerase Rpb1, domain 2 / RNA polymerase Rpb1, domain 5 / RNA polymerase Rpb1, domain 4 / DNA-directed RNA polymerase, insert domain superfamily / RNA polymerase Rpb2, domain 2 / RNA polymerase Rpb2, domain 2 / RNA polymerase, N-terminal / RNA polymerase Rpb1, funnel domain superfamily / 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 / Winged helix-like DNA-binding domain superfamily
Similarity search - Domain/homology
DNA / DNA (> 10) / DNA-directed RNA polymerase subunit alpha / DNA-directed RNA polymerase subunit beta' / RNA polymerase sigma factor / Probable DNA-directed RNA polymerase subunit delta / DNA-directed RNA polymerase subunit epsilon / DNA-directed RNA polymerase subunit beta / DNA-directed RNA polymerase subunit omega
Similarity search - Component
Biological speciesStaphylococcus aureus (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsYuan, L. / Xu, L. / Liu, Q. / Feng, Y.
Funding support China, 1items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC) China
CitationJournal: Nat Commun / Year: 2024
Title: Structural basis of promoter recognition by Staphylococcus aureus RNA polymerase.
Authors: Linggang Yuan / Qingyang Liu / Liqiao Xu / Bing Wu / Yu Feng /
Abstract: Bacterial RNAP needs to form holoenzyme with σ factors to initiate transcription. While Staphylococcus aureus σ controls housekeeping functions, S. aureus σ regulates virulence, biofilm formation, ...Bacterial RNAP needs to form holoenzyme with σ factors to initiate transcription. While Staphylococcus aureus σ controls housekeeping functions, S. aureus σ regulates virulence, biofilm formation, persistence, cell internalization, membrane transport, and antimicrobial resistance. Besides the sequence difference, the spacers between the -35 element and -10 element of σ regulated promoters are shorter than those of σ regulated promoters. Therefore, how σ recognizes and initiates transcription from target promoters can not be inferred from that of the well studied σ. Here, we report the cryo-EM structures of S. aureus RNAP-promoter open complexes comprising σ and σ, respectively. Structural analyses, in combination with biochemical experiments, reveal the structural basis for the promoter specificity of S. aureus transcription. Although the -10 element of σ regulated promoters is recognized by domain σ as single-stranded DNA, the -10 element of σ regulated promoters is co-recognized by domains σ and σ as double-stranded DNA, accounting for the short spacers of σ regulated promoters. S. aureus RNAP is a validated target of antibiotics, and our structures pave the way for rational drug design targeting S. aureus RNAP.
History
DepositionNov 21, 2023Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Jun 5, 2024Provider: repository / Type: Initial release
Revision 1.1Jun 19, 2024Group: 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_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID

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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: Probable DNA-directed RNA polymerase subunit delta
F: DNA-directed RNA polymerase subunit omega
G: DNA-directed RNA polymerase subunit epsilon
H: RNA polymerase sigma factor
T: DNA (70-mer)
N: DNA (70-mer)


Theoretical massNumber of molelcules
Total (without water)449,50610
Polymers449,50610
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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DNA-directed RNA polymerase subunit ... , 5 types, 6 molecules ABCDFG

#1: Protein DNA-directed RNA polymerase subunit alpha


Mass: 35052.656 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Staphylococcus aureus (bacteria) / Gene: rpoA / Production host: Escherichia coli (E. coli) / References: UniProt: A0A0D1GTM7
#2: Protein DNA-directed RNA polymerase subunit beta


Mass: 133381.312 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Staphylococcus aureus (bacteria) / Gene: rpoB / Production host: Escherichia coli (E. coli) / References: UniProt: W8UT31
#3: Protein DNA-directed RNA polymerase subunit beta'


Mass: 135594.266 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Staphylococcus aureus (bacteria) / Gene: rpoC / Production host: Escherichia coli (E. coli) / References: UniProt: A0A2C6P019
#5: Protein DNA-directed RNA polymerase subunit omega


Mass: 8161.236 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Staphylococcus aureus (bacteria) / Gene: rpoZ / Production host: Escherichia coli (E. coli) / References: UniProt: W8UYF2
#6: Protein DNA-directed RNA polymerase subunit epsilon


Mass: 8764.739 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Staphylococcus aureus (bacteria) / Gene: rnpZA / Production host: Escherichia coli (E. coli) / References: UniProt: A0A7I0YN47

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Protein , 2 types, 2 molecules EH

#4: Protein Probable DNA-directed RNA polymerase subunit delta


Mass: 20893.010 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Staphylococcus aureus (bacteria) / Gene: rpoE / Production host: Escherichia coli (E. coli) / References: UniProt: A0A7I0YLU8
#7: Protein RNA polymerase sigma factor


Mass: 29474.559 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Staphylococcus aureus (bacteria) / Gene: sigB / Production host: Escherichia coli (E. coli) / References: UniProt: A0A390QYZ6

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DNA chain , 2 types, 2 molecules TN

#8: DNA chain DNA (70-mer) / template strand DNA


Mass: 21360.715 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Staphylococcus aureus (bacteria)
#9: DNA chain DNA (70-mer)


Mass: 21771.047 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Staphylococcus aureus (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: sigB-dependent RNAP-promoter open complex / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Source (natural)Organism: Staphylococcus aureus (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
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: 51 e/Å2 / Film or detector model: FEI FALCON IV (4k 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: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 95089 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00327891
ELECTRON MICROSCOPYf_angle_d0.5538020
ELECTRON MICROSCOPYf_dihedral_angle_d16.2864334
ELECTRON MICROSCOPYf_chiral_restr0.0424288
ELECTRON MICROSCOPYf_plane_restr0.0054687

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