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- PDB-6n4c: EM structure of the DNA wrapping in bacterial open transcription ... -

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Basic information

Entry
Database: PDB / ID: 6n4c
TitleEM structure of the DNA wrapping in bacterial open transcription initiation complex
Components
  • (DNA (94-MER)) x 2
  • (DNA-directed RNA polymerase subunit ...Polymerase) x 5
  • RNA polymerase sigma factor RpoD
Keywordstranscription/dna / DNA wrapping / bacterial transcription initiation complex / transmission electron microscopy / single particle analysis. / TRANSCRIPTION / transcription-dna complex
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 / DNA-directed RNA polymerase 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 / protein dimerization activity / response to antibiotic / negative regulation of DNA-templated transcription / DNA-templated transcription / magnesium ion binding / DNA binding / zinc ion binding / membrane / cytosol / cytoplasm
Similarity search - Function
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 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 / Winged helix-like DNA-binding domain superfamily
Similarity search - Domain/homology
DNA / DNA (> 10) / DNA-directed RNA polymerase subunit beta / DNA-directed RNA polymerase subunit beta' / 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
Similarity search - Component
Biological speciesEscherichia coli K-12 (bacteria)
Enterobacteria phage lambda (virus)
MethodELECTRON MICROSCOPY / single particle reconstruction / negative staining / Resolution: 17 Å
AuthorsFlorez-Ariza, A. / Cassago, A. / de Oliveira, P.S.L. / Guerra, D.G. / Portugal, R.V.
CitationJournal: Biorxiv / Year: 2020
Title: Interactions of Upstream and Downstream Promoter Regions with RNA Polymerase are Energetically Coupled and a Target of Regulation in Transcription Initiation
Authors: Sosa, R. / Florez-Ariza, A. / Diaz-Celis, C. / Onoa, B. / Cassago, A. / de Oliveira, P.S.L. / Portugal, R.V. / Guerra, D.G. / Bustamante, C.
History
DepositionNov 19, 2018Deposition site: RCSB / Processing site: RCSB
Revision 1.0May 27, 2020Provider: repository / Type: Initial release

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Structure visualization

Movie
  • Deposited structure unit
  • Imaged by Jmol
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  • Superimposition on EM map
  • EMDB-0340
  • Imaged by UCSF Chimera
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Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
F: RNA polymerase sigma factor RpoD
C: DNA-directed RNA polymerase subunit beta
D: DNA-directed RNA polymerase subunit beta'
A: DNA-directed RNA polymerase subunit alpha
B: DNA-directed RNA polymerase subunit alpha
E: DNA-directed RNA polymerase subunit omega
a: DNA (94-MER)
b: DNA (94-MER)


Theoretical massNumber of molelcules
Total (without water)503,1118
Polymers503,1118
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: native gel electrophoresis, microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein , 1 types, 1 molecules F

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


Mass: 64322.277 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli K-12 (bacteria) / Strain: K12 / References: UniProt: P00579

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DNA-directed RNA polymerase subunit ... , 5 types, 5 molecules CDABE

#2: Protein DNA-directed RNA polymerase subunit beta / Polymerase / RNAP subunit beta / RNA polymerase subunit beta / Transcriptase subunit beta


Mass: 150689.672 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli K-12 (bacteria) / Strain: K12
References: UniProt: A0A0A0GWV9, UniProt: P0A8V2*PLUS, DNA-directed RNA polymerase
#3: Protein DNA-directed RNA polymerase subunit beta' / Polymerase / RNAP subunit beta' / RNA polymerase subunit beta' / Transcriptase subunit beta'


Mass: 150320.219 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli K-12 (bacteria) / Strain: K12
References: UniProt: A0A369F490, UniProt: P0A8T7*PLUS, DNA-directed RNA polymerase
#4: Protein DNA-directed RNA polymerase subunit alpha / Polymerase / RNAP subunit alpha / RNA polymerase subunit alpha / Transcriptase subunit alpha


Mass: 35275.273 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli K-12 (bacteria) / Strain: K12 / References: UniProt: P0A7Z4, DNA-directed RNA polymerase
#5: Protein DNA-directed RNA polymerase subunit alpha / Polymerase / RNAP subunit alpha / RNA polymerase subunit alpha / Transcriptase subunit alpha


Mass: 34400.266 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli K-12 (bacteria) / Strain: K12 / References: UniProt: P0A7Z4, DNA-directed RNA polymerase
#6: Protein DNA-directed RNA polymerase subunit omega / Polymerase / RNAP omega subunit / RNA polymerase omega subunit / Transcriptase subunit omega


Mass: 10118.352 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli K-12 (bacteria) / Strain: K12 / References: UniProt: P0A800, DNA-directed RNA polymerase

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DNA chain , 2 types, 2 molecules ab

#7: DNA chain DNA (94-MER)


Mass: 29094.617 Da / Num. of mol.: 1 / Source method: obtained synthetically / Details: NT strand DNA (94-MER) / Source: (synth.) Enterobacteria phage lambda (virus)
#8: DNA chain DNA (94-MER)


Mass: 28890.529 Da / Num. of mol.: 1 / Source method: obtained synthetically / Details: T strand DNA (94-MER) / Source: (synth.) Enterobacteria phage lambda (virus)

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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: E. coli RNAP-DNA wrapped open complex / Type: COMPLEX
Details: Wrapped transcription initiation open complex assembled between E. coli RNAP-sigma 70 holoenzyme and lambda PR wild-type promoter (+18 to -76)
Entity ID: all / Source: MULTIPLE SOURCES
Molecular weightValue: 0.50 MDa / Experimental value: NO
Source (natural)Organism: Escherichia coli BL21 (bacteria)
Buffer solutionpH: 7.9
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: YES / Vitrification applied: NO
EM stainingType: NEGATIVE
Details: Negatively stained EM specimens were prepared using a 2% uranyl acetate solution.
Material: Uranyl Acetate
Specimen supportDetails: na

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Electron microscopy imaging

MicroscopyModel: JEOL 2100
Electron gunElectron source: LAB6 / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy
Image recordingElectron dose: 20 e/Å2 / Film or detector model: TVIPS TEMCAM-F416 (4k x 4k)

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Processing

EM software
IDNameCategory
4IMAGICCTF correction
7UCSF Chimeramodel fitting
9IMAGICinitial Euler assignment
10IMAGICfinal Euler assignment
11IMAGICclassification
12IMAGIC3D reconstruction
CTF correctionType: PHASE FLIPPING ONLY
Particle selectionNum. of particles selected: 60393
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 17 Å / Resolution method: FSC 1/2 BIT CUT-OFF / Num. of particles: 16015 / Symmetry type: POINT
Atomic model buildingProtocol: OTHER
Details: Initial fiting was done using manual fitting in Chimera. Yasara software was used for energy minimizaton of the DNA and RNAP coordinates model.
Atomic model buildingPDB-ID: 4YLN

4yln

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