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- PDB-7xl4: Cryo-EM structure of Pseudomonas aeruginosa RNAP sigmaS holoenzym... -

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Basic information

Entry
Database: PDB / ID: 7xl4
TitleCryo-EM structure of Pseudomonas aeruginosa RNAP sigmaS holoenzyme complexes with transcription factor SutA (closed lobe)
Components
  • (DNA-directed RNA polymerase subunit ...) x 4
  • RNA polymerase sigma factor RpoS
  • Transcriptional factor SutA
KeywordsTRANSCRIPTION / transcription initiation / Pseudomonas aeruginosa / RNA polymerase / sigmaS / SutA / RNAP beta lobe / closed beta lobe
Function / homology
Function and homology information


regulation of response to salt stress / negative regulation of secondary metabolite biosynthetic process / positive regulation of single-species biofilm formation on inanimate substrate / regulation of cellular response to oxidative stress / positive regulation of chemotaxis / regulation of cell motility / sigma factor activity / bacterial-type RNA polymerase core enzyme binding / cytosolic DNA-directed RNA polymerase complex / regulation of cellular response to heat ...regulation of response to salt stress / negative regulation of secondary metabolite biosynthetic process / positive regulation of single-species biofilm formation on inanimate substrate / regulation of cellular response to oxidative stress / positive regulation of chemotaxis / regulation of cell motility / sigma factor activity / bacterial-type RNA polymerase core enzyme binding / cytosolic DNA-directed RNA polymerase complex / regulation of cellular response to heat / 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
: / : / SutA RNAP-binding domain / RNA polymerase sigma factor RpoS / RNA polymerase II, Rpb2 subunit, wall domain / RNA polymerase subunit, RPB6/omega / Eukaryotic RPB6 RNA polymerase subunit / Sigma-70 factors family signature 1. / : / RNA polymerase sigma-70 region 1.2 ...: / : / SutA RNAP-binding domain / RNA polymerase sigma factor RpoS / RNA polymerase II, Rpb2 subunit, wall domain / RNA polymerase subunit, RPB6/omega / Eukaryotic RPB6 RNA polymerase subunit / Sigma-70 factors family signature 1. / : / 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 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 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 / 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-directed RNA polymerase subunit alpha / RNA polymerase sigma factor RpoS / DNA-directed RNA polymerase subunit beta / DNA-directed RNA polymerase subunit omega / Transcriptional regulator SutA RNAP-binding domain-containing protein / DNA-directed RNA polymerase subunit beta'
Similarity search - Component
Biological speciesPseudomonas aeruginosa PAO1 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.86 Å
AuthorsHe, D.W. / You, L.L. / Zhang, Y.
Funding support China, 2items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC)31670067 China
National Natural Science Foundation of China (NSFC)31822001 China
CitationJournal: Nat Commun / Year: 2022
Title: Pseudomonas aeruginosa SutA wedges RNAP lobe domain open to facilitate promoter DNA unwinding.
Authors: Dingwei He / Linlin You / Xiaoxian Wu / Jing Shi / Aijia Wen / Zhi Yan / Wenhui Mu / Chengli Fang / Yu Feng / Yu Zhang /
Abstract: Pseudomonas aeruginosa (Pae) SutA adapts bacteria to hypoxia and nutrition-limited environment during chronic infection by increasing transcription activity of an RNA polymerase (RNAP) holoenzyme ...Pseudomonas aeruginosa (Pae) SutA adapts bacteria to hypoxia and nutrition-limited environment during chronic infection by increasing transcription activity of an RNA polymerase (RNAP) holoenzyme comprising the stress-responsive σ factor σ (RNAP-σ). SutA shows no homology to previously characterized RNAP-binding proteins. The structure and mode of action of SutA remain unclear. Here we determined cryo-EM structures of Pae RNAP-σ holoenzyme, Pae RNAP-σ holoenzyme complexed with SutA, and Pae RNAP-σ transcription initiation complex comprising SutA. The structures show SutA pinches RNAP-β protrusion and facilitates promoter unwinding by wedging RNAP-β lobe open. Our results demonstrate that SutA clears an energetic barrier to facilitate promoter unwinding of RNAP-σ holoenzyme.
History
DepositionApr 21, 2022Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Jul 27, 2022Provider: repository / Type: Initial release
Revision 1.1Feb 8, 2023Group: 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
Revision 1.2Jul 3, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond / em_admin / Item: _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
G: Transcriptional factor SutA
F: RNA polymerase sigma factor RpoS
hetero molecules


Theoretical massNumber of molelcules
Total (without water)443,83510
Polymers443,6807
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, 5 molecules ABCDE

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


Mass: 38264.258 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pseudomonas aeruginosa PAO1 (bacteria)
Strain: ATCC 15692 / DSM 22644 / CIP 104116 / JCM 14847 / LMG 12228 / 1C / PRS 101 / PAO1
Gene: rpoA, PA4238 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: O52760, DNA-directed RNA polymerase
#2: Protein DNA-directed RNA polymerase subunit beta / RNAP subunit beta / RNA polymerase subunit beta / Transcriptase subunit beta


Mass: 151225.297 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pseudomonas aeruginosa PAO1 (bacteria)
Strain: ATCC 15692 / DSM 22644 / CIP 104116 / JCM 14847 / LMG 12228 / 1C / PRS 101 / PAO1
Gene: rpoB, PA4270 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q51561, DNA-directed RNA polymerase
#3: Protein DNA-directed RNA polymerase subunit beta' / RNAP subunit beta' / RNA polymerase subunit beta' / Transcriptase subunit beta'


Mass: 156038.516 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pseudomonas aeruginosa PAO1 (bacteria)
Strain: ATCC 15692 / DSM 22644 / CIP 104116 / JCM 14847 / LMG 12228 / 1C / PRS 101 / PAO1
Gene: rpoC, PA4269 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q9HWC9, DNA-directed RNA polymerase
#4: Protein DNA-directed RNA polymerase subunit omega / RNAP omega subunit / RNA polymerase omega subunit / Transcriptase subunit omega


Mass: 9783.876 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pseudomonas aeruginosa PAO1 (bacteria)
Strain: ATCC 15692 / DSM 22644 / CIP 104116 / JCM 14847 / LMG 12228 / 1C / PRS 101 / PAO1
Gene: rpoZ, PA5337 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q9HTM1, DNA-directed RNA polymerase

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Protein , 2 types, 2 molecules GF

#5: Protein Transcriptional factor SutA


Mass: 11497.365 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pseudomonas aeruginosa PAO1 (bacteria)
Strain: ATCC 15692 / DSM 22644 / CIP 104116 / JCM 14847 / LMG 12228 / 1C / PRS 101 / PAO1
Gene: PA5285 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q9HTR9
#6: Protein RNA polymerase sigma factor RpoS / Sigma S / Sigma-38


Mass: 38606.742 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pseudomonas aeruginosa PAO1 (bacteria)
Strain: ATCC 15692 / DSM 22644 / CIP 104116 / JCM 14847 / LMG 12228 / 1C / PRS 101 / PAO1
Gene: rpoS, PA3622 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P45684

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Non-polymers , 2 types, 3 molecules

#7: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Zn
#8: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg

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Details

Has ligand of interestN

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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: Pseudomonas aeruginosa RNAP sigmaS holoenzyme with transcription factor SutA complex(closed lobe)
Type: COMPLEX / Entity ID: #1-#6 / Source: RECOMBINANT
Molecular weightValue: 0.433 MDa / Experimental value: YES
Source (natural)Organism: Pseudomonas aeruginosa PAO1 (bacteria)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 7.5
SpecimenConc.: 15 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationCryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 283 K / Details: blot 4 seconds before plunging

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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: 64000 X / Nominal defocus max: 2400 nm / Nominal defocus min: 1200 nm / Cs: 0 mm
Specimen holderCryogen: NITROGEN
Image recordingElectron dose: 50 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K3 (6k x 4k)

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Processing

EM software
IDNameVersionCategory
2EPUimage acquisition
4CTFFIND4CTF correction
10RELION3.08initial Euler assignment
11RELION3.08final Euler assignment
12RELION3.08classification
13RELION3.083D reconstruction
CTF correctionType: NONE
3D reconstructionResolution: 3.86 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 29531 / Symmetry type: POINT

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