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- PDB-7bef: Structures of class II bacterial transcription complexes -

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Basic information

Entry
Database: PDB / ID: 7bef
TitleStructures of class II bacterial transcription complexes
Components
  • (DNA-directed RNA polymerase subunit ...Polymerase) x 4
  • (pmicF promoter ...) x 2
  • RNA polymerase sigma factor RpoD
  • Transcriptional activator RamA
KeywordsTRANSCRIPTION / antibiotic resistance / bacterial transcription activator / cryoEM
Function / homology
Function and homology information


sigma factor antagonist complex / RNA polymerase complex / submerged biofilm formation / cellular response to cell envelope stress / cytosolic DNA-directed RNA polymerase complex / regulation of DNA-templated transcription initiation / bacterial-type flagellum assembly / sigma factor activity / bacterial-type flagellum-dependent cell motility / nitrate assimilation ...sigma factor antagonist complex / RNA polymerase complex / submerged biofilm formation / cellular response to cell envelope stress / cytosolic DNA-directed RNA polymerase complex / regulation of DNA-templated transcription initiation / bacterial-type flagellum assembly / sigma factor activity / bacterial-type flagellum-dependent cell motility / nitrate assimilation / transcription elongation factor complex / regulation of DNA-templated transcription elongation / transcription antitermination / cell motility / DNA-templated transcription initiation / ribonucleoside binding / DNA-directed 5'-3' RNA polymerase activity / DNA-directed RNA polymerase / response to heat / protein-containing complex assembly / intracellular iron ion homeostasis / sequence-specific DNA binding / protein dimerization activity / DNA-binding transcription factor activity / response to antibiotic / negative regulation of DNA-templated transcription / magnesium ion binding / DNA binding / zinc ion binding / membrane / cytosol / cytoplasm
Similarity search - Function
Transcription regulator HTH, AraC- type / HTH domain AraC-type, conserved site / Bacterial regulatory proteins, araC family signature. / DNA binding HTH domain, AraC-type / Helix-turn-helix domain / 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 ...Transcription regulator HTH, AraC- type / HTH domain AraC-type, conserved site / Bacterial regulatory proteins, araC family signature. / DNA binding HTH domain, AraC-type / Helix-turn-helix domain / 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 / 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, 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 Rpb1, domain 3 / RNA polymerase Rpb1, domain 3 / RNA polymerase Rpb2, domain 2 superfamily / RNA polymerase Rpb1, domain 1 / RNA polymerase Rpb1, domain 1 / RNA polymerase, alpha subunit / RNA polymerase Rpb1, domain 4 / RNA polymerase Rpb1, domain 2 / RNA polymerase Rpb1, domain 4 / RNA polymerase, N-terminal / RNA polymerase Rpb1, funnel domain superfamily / RNA polymerase I subunit A N-terminus / RNA polymerase Rpb1, domain 5 / RNA polymerase Rpb1, domain 5 / RNA polymerase, beta subunit, protrusion / RNA polymerase beta subunit / 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 / Homeobox-like domain superfamily / Winged helix-like DNA-binding domain superfamily
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 / Transcriptional activator RamA
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
Klebsiella pneumoniae (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.5 Å
AuthorsHao, M. / Ye, F.Z. / Zhang, X.D.
Funding support United Kingdom, China, 3items
OrganizationGrant numberCountry
Biotechnology and Biological Sciences Research Council (BBSRC)BB/N007816/1 United Kingdom
Medical Research Council (MRC, United Kingdom)MR/P007503/1 United Kingdom
National Science Foundation (NSF, China)81991531 China
CitationJournal: Adv Sci (Weinh) / Year: 2022
Title: Structures of Class I and Class II Transcription Complexes Reveal the Molecular Basis of RamA-Dependent Transcription Activation.
Authors: Min Hao / Fuzhou Ye / Milija Jovanovic / Ioly Kotta-Loizou / Qingqing Xu / Xiaohua Qin / Martin Buck / Xiaodong Zhang / Minggui Wang /
Abstract: Transcription activator RamA is linked to multidrug resistance of Klebsiella pneumoniae through controlling genes that encode efflux pumps (acrA) and porin-regulating antisense RNA (micF). In ...Transcription activator RamA is linked to multidrug resistance of Klebsiella pneumoniae through controlling genes that encode efflux pumps (acrA) and porin-regulating antisense RNA (micF). In bacteria, σ , together with activators, controls the majority of genes by recruiting RNA polymerase (RNAP) to the promoter regions. RNAP and σ form a holoenzyme that recognizes -35 and -10 promoter DNA consensus sites. Many activators bind upstream from the holoenzyme and can be broadly divided into two classes. RamA acts as a class I activator on acrA and class II activator on micF, respectively. The authors present biochemical and structural data on RamA in complex with RNAP-σ at the two promoters and the data reveal the molecular basis for how RamA assembles and interacts with core RNAP and activates transcription that contributes to antibiotic resistance. Further, comparing with CAP/TAP complexes reveals common and activator-specific features in activator binding and uncovers distinct roles of the two C-terminal domains of RNAP α subunit.
History
DepositionDec 23, 2020Deposition site: PDBE / Processing site: PDBE
Revision 1.0Dec 1, 2021Provider: repository / Type: Initial release
Revision 1.1Feb 16, 2022Group: Database references / Category: citation / Item: _citation.journal_volume / _citation.year

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Structure visualization

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  • Deposited structure unit
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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
G: Transcriptional activator RamA
T: pmicF promoter template DNA
N: pmicF promoter non-template DNA


Theoretical massNumber of molelcules
Total (without water)522,4759
Polymers522,4759
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area60300 Å2
ΔGint-283 kcal/mol
Surface area202680 Å2
MethodPISA

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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 / Polymerase / 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 (strain K12) (bacteria)
Strain: K12 / Gene: rpoA, pez, phs, sez, b3295, JW3257 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P0A7Z4, DNA-directed RNA polymerase
#2: Protein DNA-directed RNA polymerase subunit beta / Polymerase / RNAP subunit beta / RNA polymerase subunit beta / Transcriptase subunit beta


Mass: 150820.875 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (strain K12) (bacteria)
Strain: K12
Gene: rpoB, groN, nitB, rif, ron, stl, stv, tabD, b3987, JW3950
Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P0A8V2, DNA-directed RNA polymerase
#3: Protein DNA-directed RNA polymerase subunit beta' / Polymerase / 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)
Strain: K12 / Gene: rpoC, tabB, b3988, JW3951 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P0A8T7, DNA-directed RNA polymerase
#4: Protein DNA-directed RNA polymerase subunit omega / Polymerase / 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)
Strain: K12 / Gene: rpoZ, b3649, JW3624 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P0A800, DNA-directed RNA polymerase

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Protein , 2 types, 2 molecules FG

#5: Protein RNA polymerase sigma factor RpoD / Sigma-70


Mass: 72344.500 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (strain K12) (bacteria)
Strain: K12 / Gene: rpoD, alt, b3067, JW3039 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P00579
#6: Protein Transcriptional activator RamA


Mass: 15510.520 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Klebsiella pneumoniae (bacteria) / Gene: ramA / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q48413

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PmicF promoter ... , 2 types, 2 molecules TN

#7: DNA chain pmicF promoter template DNA


Mass: 22584.451 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Klebsiella pneumoniae (bacteria) / Production host: Escherichia coli BL21(DE3) (bacteria)
#8: DNA chain pmicF promoter non-template DNA


Mass: 22480.471 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Klebsiella pneumoniae (bacteria) / Production host: Escherichia coli BL21(DE3) (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

Component
IDNameTypeEntity IDParent-IDSource
1Class II transcription complexCOMPLEXall0MULTIPLE SOURCES
2DNA-directed RNA polymerasePolymeraseCOMPLEX#1-#51RECOMBINANT
3Transcriptional activator RamA and pmicF promoter DNAsCOMPLEX#6-#81RECOMBINANT
Molecular weightValue: 0.56 MDa / Experimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Escherichia coli K-12 (bacteria)83333
33Klebsiella pneumoniae (bacteria)573
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
22Escherichia coli BL21(DE3) (bacteria)469008
33Escherichia coli BL21(DE3) (bacteria)469008
Buffer solutionpH: 8 / Details: 20 mM Tris pH 8, 50 mM NaCl
Buffer component
IDConc.FormulaBuffer-ID
120 mMTRIS1
250 mMNaClSodium chloride1
SpecimenConc.: 0.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 100 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: OTHER
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 75000 X / Cs: 2.7 mm / C2 aperture diameter: 70 µm
Specimen holderSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 79.1 e/Å2 / Film or detector model: FEI FALCON III (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 2878

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Processing

EM software
IDNameCategory
4GctfCTF correction
7UCSF Chimeramodel fitting
9RELIONinitial Euler assignment
10RELIONfinal Euler assignment
13Cootmodel refinement
14PHENIXmodel refinement
15REFMACmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 774955
3D reconstructionResolution: 4.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 74282 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Atomic model buildingPDB-ID: 4YLP

4ylp

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