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- PDB-8gzg: Cryo-EM structure of Synechocystis sp. PCC 6803 RPitc -

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Basic information

Entry
Database: PDB / ID: 8gzg
TitleCryo-EM structure of Synechocystis sp. PCC 6803 RPitc
Components
  • (DNA-directed RNA polymerase subunit ...) x 5
  • Nontemplate strand DNA
  • RNA (5'-R(*UP*CP*GP*A)-3')
  • RNA polymerase sigma factor SigA
  • Template strand DNA
KeywordsTRANSCRIPTION/DNA/RNA / Cryo-EM / Synechocystis sp. PCC 6803 / Syn6803 / Transcription initiation / RPitc / Transcription / Cyanobacteria / TRANSCRIPTION-DNA-RNA complex
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 / magnesium ion binding / DNA 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 / magnesium ion binding / DNA binding / zinc ion binding / cytoplasm
Similarity search - Function
RNA polymerase sigma factor, RpoD-like, cyanobacteria / DNA-directed RNA polymerase, subunit gamma / DNA-directed RNA polymerase subunit RpoC1 / DNA-directed RNA polymerase, subunit beta'' / Dna-directed Rna Polymerase Ii 140kd Polypeptide; Chain: B; domain 3 / RNA polymerase Rpb2, domain 2 / RNA polymerase II, Rpb2 subunit, wall domain / Sigma-70 factors family signature 1. / RNA polymerase sigma factor RpoD, C-terminal / RNA polymerase sigma factor RpoD ...RNA polymerase sigma factor, RpoD-like, cyanobacteria / DNA-directed RNA polymerase, subunit gamma / DNA-directed RNA polymerase subunit RpoC1 / DNA-directed RNA polymerase, subunit beta'' / Dna-directed Rna Polymerase Ii 140kd Polypeptide; Chain: B; domain 3 / RNA polymerase Rpb2, domain 2 / RNA polymerase II, Rpb2 subunit, wall domain / 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, 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 / DNA-directed RNA polymerase, subunit beta-prime / 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 / OB fold (Dihydrolipoamide Acetyltransferase, E2P) / Winged helix-like DNA-binding domain superfamily / Alpha-Beta Complex / Beta Barrel / Mainly Beta / Alpha Beta
Similarity search - Domain/homology
DNA / DNA (> 10) / RNA / DNA-directed RNA polymerase subunit alpha / DNA-directed RNA polymerase subunit beta' / DNA-directed RNA polymerase subunit gamma / DNA-directed RNA polymerase subunit omega / RNA polymerase sigma factor SigA / DNA-directed RNA polymerase subunit beta
Similarity search - Component
Biological speciesSynechocystis sp. PCC 6803 (bacteria)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.13 Å
AuthorsShen, L.Q. / You, L.L. / Zhang, Y.
Funding support China, 1items
OrganizationGrant numberCountry
Chinese Academy of Sciences China
CitationJournal: Proc Natl Acad Sci U S A / Year: 2023
Title: An SI3-σ arch stabilizes cyanobacteria transcription initiation complex.
Authors: Liqiang Shen / Giorgio Lai / Linlin You / Jing Shi / Xiaoxian Wu / Maria Puiu / Zhanxi Gu / Yu Feng / Yulia Yuzenkova / Yu Zhang /
Abstract: Multisubunit RNA polymerases (RNAPs) associate with initiation factors (σ in bacteria) to start transcription. The σ factors are responsible for recognizing and unwinding promoter DNA in all ...Multisubunit RNA polymerases (RNAPs) associate with initiation factors (σ in bacteria) to start transcription. The σ factors are responsible for recognizing and unwinding promoter DNA in all bacterial RNAPs. Here, we report two cryo-EM structures of cyanobacterial transcription initiation complexes at near-atomic resolutions. The structures show that cyanobacterial RNAP forms an "SI3-σ" arch interaction between domain 2 of σ (σ) and sequence insertion 3 (SI3) in the mobile catalytic domain Trigger Loop (TL). The "SI3-σ" arch facilitates transcription initiation from promoters of different classes through sealing the main cleft and thereby stabilizing the RNAP-promoter DNA open complex. Disruption of the "SI3-σ" arch disturbs cyanobacteria growth and stress response. Our study reports the structure of cyanobacterial RNAP and a unique mechanism for its transcription initiation. Our data suggest functional plasticity of SI3 and provide the foundation for further research into cyanobacterial and chloroplast transcription.
History
DepositionSep 27, 2022Deposition site: PDBJ / Processing site: RCSB
Revision 1.0Apr 19, 2023Provider: repository / Type: Initial release
Revision 1.1Jun 19, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond
Revision 1.2Oct 30, 2024Group: Data collection / Database references / Structure summary
Category: citation / citation_author ...citation / citation_author / em_admin / pdbx_entry_details
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 / _em_admin.last_update / _pdbx_entry_details.has_protein_modification

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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 gamma
E: DNA-directed RNA polymerase subunit omega
F: RNA polymerase sigma factor SigA
Z: DNA-directed RNA polymerase subunit beta'
1: Nontemplate strand DNA
2: Template strand DNA
3: RNA (5'-R(*UP*CP*GP*A)-3')
hetero molecules


Theoretical massNumber of molelcules
Total (without water)515,44413
Polymers515,28910
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 ... , 5 types, 6 molecules ABCDEZ

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


Mass: 36657.344 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Synechocystis sp. PCC 6803 (bacteria) / Strain: PCC 6803 / Kazusa / Gene: rpoA, sll1818 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P73297, DNA-directed RNA polymerase
#2: Protein DNA-directed RNA polymerase subunit beta / RNAP subunit beta / RNA polymerase subunit beta / Transcriptase subunit beta


Mass: 123719.812 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Synechocystis sp. PCC 6803 (bacteria) / Strain: PCC 6803 / Kazusa / Gene: rpoB, sll1787 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P77965, DNA-directed RNA polymerase
#3: Protein DNA-directed RNA polymerase subunit gamma / RNAP subunit gamma / RNA polymerase subunit gamma / Transcriptase subunit gamma


Mass: 71072.992 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Synechocystis sp. PCC 6803 (bacteria) / Strain: PCC 6803 / Kazusa / Gene: rpoC1, slr1265 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P74177, DNA-directed RNA polymerase
#4: Protein DNA-directed RNA polymerase subunit omega / RNAP omega subunit / RNA polymerase omega subunit / Transcriptase subunit omega


Mass: 8749.776 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Synechocystis sp. PCC 6803 (bacteria) / Strain: PCC 6803 / Kazusa / Gene: rpoZ, ssl2982 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P74352, DNA-directed RNA polymerase
#6: Protein DNA-directed RNA polymerase subunit beta' / RNAP subunit beta' / RNA polymerase subunit beta' / Transcriptase subunit beta'


Mass: 145371.984 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Synechocystis sp. PCC 6803 (bacteria) / Strain: PCC 6803 / Kazusa / Gene: rpoC2, sll1789 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P73334, DNA-directed RNA polymerase

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DNA chain , 2 types, 2 molecules 12

#7: DNA chain Nontemplate strand DNA


Mass: 20810.250 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#8: DNA chain Template strand DNA


Mass: 20673.232 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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Protein / RNA chain , 2 types, 2 molecules F3

#5: Protein RNA polymerase sigma factor SigA


Mass: 50335.371 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Synechocystis sp. PCC 6803 (bacteria) / Strain: PCC 6803 / Kazusa / Gene: sigA, rpoD, rpoD1, slr0653 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P74565
#9: RNA chain RNA (5'-R(*UP*CP*GP*A)-3')


Mass: 1240.802 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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Non-polymers , 2 types, 3 molecules

#10: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg
#11: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Zn

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Details

Has ligand of interestN
Has protein modificationN

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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: The complex of Synechocystis sp. PCC 6803 RPitc / Type: COMPLEX / Entity ID: #1-#9 / Source: MULTIPLE SOURCES
Molecular weightValue: 0.5 MDa / Experimental value: YES
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
21Synechocystis sp. PCC 6803 (bacteria)1148
31synthetic construct (others)32630
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 7.5
SpecimenConc.: 10 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid type: C-flat-1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 283 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: 2600 nm / Nominal defocus min: 1800 nm
Image recordingElectron dose: 53.6 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 3432

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Processing

EM software
IDNameVersionCategory
1RELION3.08particle selection
2SerialEMimage acquisition
4RELION3.08CTF correction
7UCSF Chimera10.14model fitting
9RELION3.08initial Euler assignment
10RELIONfinal Euler assignment
11RELION3.08classification
12RELION3.083D reconstruction
13PHENIX10.14model refinement
CTF correctionType: NONE
Particle selectionNum. of particles selected: 473353
3D reconstructionResolution: 3.13 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 157889 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT

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