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- PDB-7dy6: A refined cryo-EM structure of an Escherichia coli RNAP-promoter ... -

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Basic information

Entry
Database: PDB / ID: 7dy6
TitleA refined cryo-EM structure of an Escherichia coli RNAP-promoter open complex (RPo) with SspA
Components
  • (DNA (63-MER)) x 2
  • (DNA-directed RNA polymerase subunit ...) x 4
  • RNA polymerase sigma factor RpoD
  • Stringent starvation protein A
KeywordsTRANSCRIPTION / bacterial RNA polymerase / Complex
Function / homology
Function and homology information


glutathione dehydrogenase (ascorbate) activity / L-ascorbic acid metabolic process / sigma factor antagonist complex / submerged biofilm formation / cellular response to cell envelope stress / cytosolic DNA-directed RNA polymerase complex / regulation of DNA-templated transcription initiation / sigma factor activity / response to starvation / bacterial-type flagellum assembly ...glutathione dehydrogenase (ascorbate) activity / L-ascorbic acid metabolic process / sigma factor antagonist complex / submerged biofilm formation / cellular response to cell envelope stress / cytosolic DNA-directed RNA polymerase complex / regulation of DNA-templated transcription initiation / sigma factor activity / response to starvation / bacterial-type flagellum assembly / glutathione transferase activity / bacterial-type flagellum-dependent cell motility / nitrate assimilation / glutathione metabolic process / transcription elongation factor complex / regulation of DNA-templated transcription elongation / transcription antitermination / DNA-templated transcription initiation / cell motility / ribonucleoside binding / DNA-directed 5'-3' RNA polymerase activity / DNA-directed RNA polymerase / response to heat / intracellular iron ion homeostasis / protein dimerization activity / response to antibiotic / negative regulation of DNA-templated transcription / DNA-templated transcription / positive regulation of DNA-templated transcription / magnesium ion binding / DNA binding / zinc ion binding / membrane / cytoplasm / cytosol
Similarity search - Function
Stringent starvation protein A, C-terminal / Stringent starvation protein A, N-terminal / : / RNA polymerase sigma factor 70, non-essential domain / Sigma-70, non-essential region / RNA polymerase sigma factor 70, region 1.1 / Sigma-70 factor, region 1.1 superfamily / Sigma-70 factor, region 1.1 / Glutathione S-transferase, C-terminal domain / Sigma-70 factors family signature 1. ...Stringent starvation protein A, C-terminal / Stringent starvation protein A, N-terminal / : / RNA polymerase sigma factor 70, non-essential domain / Sigma-70, non-essential region / RNA polymerase sigma factor 70, region 1.1 / Sigma-70 factor, region 1.1 superfamily / Sigma-70 factor, region 1.1 / Glutathione S-transferase, C-terminal 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 / Glutathione S-transferase, N-terminal domain / Glutathione transferase family / Glutathione S-transferase, C-terminal / Soluble glutathione S-transferase N-terminal domain profile. / Glutathione S-transferase, C-terminal-like / Soluble glutathione S-transferase C-terminal domain profile. / Glutathione S-transferase, N-terminal / DNA-directed RNA polymerase, omega subunit / Glutathione S-transferase, C-terminal domain superfamily / 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 / RNA polymerase Rpb1, clamp domain superfamily / RPB6/omega subunit-like superfamily / DNA-directed RNA polymerase, subunit beta-prime / RNA polymerase Rpb2, domain 2 superfamily / RNA polymerase Rpb1, domain 3 / RNA polymerase Rpb1, domain 3 / RNA polymerase Rpb1, domain 1 / 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 / 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 / DNA-directed RNA polymerase, RpoA/D/Rpb3-type / RNA polymerase Rpb3/RpoA insert domain / RNA polymerase Rpb3/Rpb11 dimerisation domain / RNA polymerases D / 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 / Thioredoxin-like superfamily / 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 omega / RNA polymerase sigma factor RpoD / DNA-directed RNA polymerase subunit beta' / DNA-directed RNA polymerase subunit beta / Stringent starvation protein A
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.68 Å
AuthorsLin, W.
CitationJournal: Biochem Biophys Res Commun / Year: 2021
Title: A unique binding between SspA and RNAP βNTH across low-GC Gram-negative bacteria facilitates SspA-mediated transcription regulation.
Authors: Fulin Wang / Yu Feng / Zhuo Shang / Wei Lin /
Abstract: Stringent starvation protein A (SspA) involved in nucleotide metabolism, acid tolerance and virulence of bacteria has been demonstrated to function as a transcription factor to regulate σ-dependent ...Stringent starvation protein A (SspA) involved in nucleotide metabolism, acid tolerance and virulence of bacteria has been demonstrated to function as a transcription factor to regulate σ-dependent gene transcription through interacting with σ region 4 and the zinc binding domain (ZBD) of E. coli RNA polymerase (EcoRNAP) β' subunit simultaneously. Despite extensive biochemical and structural analyses were reported recently, the interactions of SspA with RNAP are not comprehensively understood. Here, we reprocessed our previous cryo-EM dataset of EcoRNAP-promoter open complex with SspA (SspA-RPo) and obtained a significantly improved density map. Unexpectedly, the new map showed that SspA interacts with both N-terminal helix of β' subunit (β'ΝΤΗ) and ω subunit, which contributes to stabilize the SspA-EcoRNAP σ holoenzyme complex. Sequence alignments and phylogenetic tree analyses of N-terminal sequences of β' subunit from different classes of bacteria revealed that β'ΝΤΗ is highly conserved and exclusively found in low-GC-content Gram-negative bacteria that harbor SspA, implying a co-evolution of β'ΝΤΗ and SspA. The transcription assays of wild-type SspA and its mutants demonstrated the interaction between SspA and β'ΝΤΗ facilitates the transcription regulation of SspA. Together, our results provide a more comprehensive insight into the interactions between SspA and RNAP and their roles in bacterial transcription regulation.
History
DepositionJan 20, 2021Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Nov 17, 2021Provider: repository / Type: Initial release
Revision 1.1Jun 5, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond

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Structure visualization

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Structure viewerMolecule:
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Assembly

Deposited unit
H: DNA (63-MER)
I: Stringent starvation protein A
J: Stringent starvation protein A
K: DNA-directed RNA polymerase subunit alpha
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'
F: RNA polymerase sigma factor RpoD
G: DNA (63-MER)
E: DNA-directed RNA polymerase subunit omega
hetero molecules


Theoretical massNumber of molelcules
Total (without water)584,10114
Polymers583,94611
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 chain , 2 types, 2 molecules HG

#1: DNA chain DNA (63-MER)


Mass: 19671.666 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Escherichia coli (E. coli)
#7: DNA chain DNA (63-MER)


Mass: 19159.285 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Escherichia coli (E. coli)

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Protein , 2 types, 3 molecules IJF

#2: Protein Stringent starvation protein A


Mass: 24332.885 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (strain K12) (bacteria)
Gene: sspA, pog, ssp, b3229, JW3198
Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
References: UniProt: P0ACA3
#6: Protein RNA polymerase sigma factor RpoD / Sigma-70


Mass: 70352.242 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (strain K12) (bacteria)
Gene: rpoD, alt, b3067, JW3039
Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
References: UniProt: P00579

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

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


Mass: 36558.680 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (strain K12) (bacteria)
Gene: rpoA, FAZ83_23195
Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
References: UniProt: A0A4S5AL01, DNA-directed RNA polymerase
#4: Protein DNA-directed RNA polymerase subunit beta / RNAP subunit beta / RNA polymerase subunit beta / Transcriptase subunit beta


Mass: 150804.922 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (strain K12) (bacteria)
Gene: rpoB, groN, nitB, rif, ron, stl, stv, tabD, b3987, JW3950
Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
References: UniProt: P0A8V2, DNA-directed RNA polymerase
#5: Protein DNA-directed RNA polymerase subunit beta' / RNAP subunit beta' / RNA polymerase subunit beta' / Transcriptase subunit beta'


Mass: 155366.781 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (strain K12) (bacteria)
Gene: rpoC, tabB, b3988, JW3951
Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
References: UniProt: P0A8T7, DNA-directed RNA polymerase
#8: Protein DNA-directed RNA polymerase subunit omega / RNAP omega subunit / RNA polymerase omega subunit / Transcriptase subunit omega


Mass: 10249.547 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (strain K12) (bacteria)
Gene: rpoZ, FAZ83_19385
Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
References: UniProt: A0A4S5AUM4, DNA-directed RNA polymerase

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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: an Escherichia coli RNAP-promoter open complex (RPo) with SspA
Type: CELL / Entity ID: #1-#8 / Source: RECOMBINANT
Source (natural)Organism: Escherichia coli (strain K12) (bacteria)
Source (recombinant)Organism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
Buffer solutionpH: 7.9
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

MicroscopyModel: FEI/PHILIPS CM300FEG/T
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: OTHER
Electron lensMode: BRIGHT FIELD
Image recordingElectron dose: 59 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.68 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 60145 / Symmetry type: POINT

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