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- PDB-7vwz: Cryo-EM structure of Rob-dependent transcription activation compl... -

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Basic information

Entry
Database: PDB / ID: 7vwz
TitleCryo-EM structure of Rob-dependent transcription activation complex in a unique conformation
Components
  • (DNA-directed RNA polymerase subunit ...) x 4
  • (micF promoter DNA ...) x 2
  • RNA polymerase sigma factor RpoD
  • Right origin-binding protein
KeywordsTRANSCRIPTION/DNA / bacterial RNA polymerase / TRANSCRIPTION-DNA COMPLEX
Function / homology
Function and homology information


bacterial-type RNA polymerase holo enzyme binding / sigma factor antagonist complex / RNA polymerase complex / submerged biofilm formation / cellular response to cell envelope stress / regulation of DNA-templated transcription initiation / sigma factor activity / bacterial-type flagellum assembly / bacterial-type RNA polymerase core enzyme binding / cytosolic DNA-directed RNA polymerase complex ...bacterial-type RNA polymerase holo enzyme binding / sigma factor antagonist complex / RNA polymerase complex / submerged biofilm formation / cellular response to cell envelope stress / regulation of DNA-templated transcription initiation / sigma factor activity / bacterial-type flagellum assembly / bacterial-type RNA polymerase core enzyme binding / cytosolic DNA-directed RNA polymerase complex / bacterial-type flagellum-dependent cell motility / nitrate assimilation / transcription elongation factor complex / regulation of DNA-templated transcription elongation / transcription antitermination / DNA-templated transcription initiation / cell motility / ribonucleoside binding / : / : / : / : / : / : / DNA-directed RNA polymerase / response to heat / protein-containing complex assembly / sequence-specific DNA binding / intracellular iron ion homeostasis / protein dimerization activity / DNA-binding transcription factor activity / response to antibiotic / negative regulation of DNA-templated transcription / regulation of DNA-templated transcription / magnesium ion binding / DNA binding / zinc ion binding / membrane / cytosol / cytoplasm
Similarity search - Function
: / Bacterial transcription activator, effector binding / Bacterial transcription activator, effector binding domain / GyrI-like small molecule binding domain / GyrI-like small molecule binding domain / Transcription regulator HTH, AraC- type / Regulatory factor, effector binding domain superfamily / HTH domain AraC-type, conserved site / Bacterial regulatory proteins, araC family signature. / Helix-turn-helix domain ...: / Bacterial transcription activator, effector binding / Bacterial transcription activator, effector binding domain / GyrI-like small molecule binding domain / GyrI-like small molecule binding domain / Transcription regulator HTH, AraC- type / Regulatory factor, effector binding domain superfamily / HTH domain AraC-type, conserved site / Bacterial regulatory proteins, araC family signature. / Helix-turn-helix domain / DNA binding HTH domain, AraC-type / Bacterial regulatory proteins, araC family DNA-binding domain profile. / helix_turn_helix, arabinose operon control protein / 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 / RNA polymerase Rpb1 funnel domain / RNA polymerase II/Efflux pump adaptor protein, barrel-sandwich hybrid domain / Sigma-70 factors family signature 1. / RNA polymerase sigma factor RpoD, C-terminal / RNA polymerase sigma factor RpoD / Topoisomerase I; Chain A, domain 4 / DNA-directed RNA polymerase, insert domain / : / 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 Alpha Subunit; Chain A, domain 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 / Beta Complex / 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 Rpb1, funnel domain superfamily / RNA polymerase I subunit A N-terminus / RNA polymerase Rpb2, domain 2 / RNA polymerase Rpb2, domain 2 / RNA polymerase, RBP11-like subunit / Homeobox-like domain superfamily / 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 / Beta Barrel / Orthogonal Bundle / Mainly Beta / Mainly Alpha
Similarity search - Domain/homology
DNA / DNA (> 10) / RNA polymerase sigma factor RpoD / DNA-directed RNA polymerase subunit alpha / DNA-directed RNA polymerase subunit omega / DNA-directed RNA polymerase subunit beta' / DNA-directed RNA polymerase subunit beta / Right origin-binding protein
Similarity search - Component
Biological speciesEscherichia coli K-12 (bacteria)
Escherichia coli (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4 Å
AuthorsLin, W. / Feng, Y. / Shi, J.
Funding support China, 1items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC) China
CitationJournal: Nucleic Acids Res / Year: 2022
Title: Structural basis of transcription activation by Rob, a pleiotropic AraC/XylS family regulator.
Authors: Jing Shi / Fulin Wang / Fangfang Li / Lu Wang / Ying Xiong / Aijia Wen / Yuanling Jin / Sha Jin / Fei Gao / Zhenzhen Feng / Jiacong Li / Yu Zhang / Zhuo Shang / Shuang Wang / Yu Feng / Wei Lin /
Abstract: Rob, which serves as a paradigm of the large AraC/XylS family transcription activators, regulates diverse subsets of genes involved in multidrug resistance and stress response. However, the ...Rob, which serves as a paradigm of the large AraC/XylS family transcription activators, regulates diverse subsets of genes involved in multidrug resistance and stress response. However, the underlying mechanism of how it engages bacterial RNA polymerase and promoter DNA to finely respond to environmental stimuli is still elusive. Here, we present two cryo-EM structures of Rob-dependent transcription activation complex (Rob-TAC) comprising of Escherichia coli RNA polymerase (RNAP), Rob-regulated promoter and Rob in alternative conformations. The structures show that a single Rob engages RNAP by interacting with RNAP αCTD and σ70R4, revealing their generally important regulatory roles. Notably, by occluding σ70R4 from binding to -35 element, Rob specifically binds to the conserved Rob binding box through its consensus HTH motifs, and retains DNA bending by aid of the accessory acidic loop. More strikingly, our ligand docking and biochemical analysis demonstrate that the large Rob C-terminal domain (Rob CTD) shares great structural similarity with the global Gyrl-like domains in effector binding and allosteric regulation, and coordinately promotes formation of competent Rob-TAC. Altogether, our structural and biochemical data highlight the detailed molecular mechanism of Rob-dependent transcription activation, and provide favorable evidences for understanding the physiological roles of the other AraC/XylS-family transcription factors.
History
DepositionNov 12, 2021Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Jun 8, 2022Provider: repository / Type: Initial release
Revision 1.1Jun 15, 2022Group: Database references / Category: citation / Item: _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.2Jun 22, 2022Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.3Jun 26, 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

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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 RpoD
1: micF promoter DNA forward strand
2: micF promoter DNA reverse strand
G: Right origin-binding protein
I: Right origin-binding protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)569,74213
Polymers569,58710
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: 36558.680 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli K-12 (bacteria) / Strain: K12 / Gene: rpoA / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / References: UniProt: P0A7Z4, DNA-directed RNA polymerase
#2: 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 K-12 (bacteria) / Strain: K12 / Gene: rpoB / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / References: UniProt: P0A8V2, DNA-directed RNA polymerase
#3: 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 K-12 (bacteria) / Strain: K12 / Gene: rpoC / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / References: UniProt: P0A8T7, DNA-directed RNA polymerase
#4: 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 K-12 (bacteria) / Strain: K12 / Gene: rpoZ / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / References: UniProt: P0A800, DNA-directed RNA polymerase

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Protein , 2 types, 3 molecules FGI

#5: 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 K-12 (bacteria) / Strain: K12 / Gene: rpoD / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / References: UniProt: P00579
#8: Protein Right origin-binding protein


Mass: 33184.844 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli K-12 (bacteria) / Strain: K12 / Gene: rob / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / References: UniProt: P0ACI0

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MicF promoter DNA ... , 2 types, 2 molecules 12

#6: DNA chain micF promoter DNA forward strand


Mass: 21574.877 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Escherichia coli (E. coli)
#7: DNA chain micF promoter DNA reverse strand


Mass: 21751.922 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Escherichia coli (E. coli)

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Non-polymers , 2 types, 3 molecules

#9: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Zn / Feature type: SUBJECT OF INVESTIGATION
#10: Chemical ChemComp-MG / MAGNESIUM ION


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

Component
IDNameTypeEntity IDParent-IDSource
1Rob-dependent transcription activation complexCOMPLEX#1-#5, #80MULTIPLE SOURCES
2RNA polymerase subunitsCOMPLEX#1-#5, #81RECOMBINANT
3DNACOMPLEX#6-#71SYNTHETIC
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
21Escherichia coli K-12 (bacteria)83333
32Escherichia coli (E. coli)562
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
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
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

CTF correctionType: NONE
3D reconstructionResolution: 4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 335021 / Symmetry type: POINT

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