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- PDB-6pst: Escherichia coli RNA polymerase promoter unwinding intermediate (... -

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Basic information

Entry
Database: PDB / ID: 6pst
TitleEscherichia coli RNA polymerase promoter unwinding intermediate (TRPi1.5b) with TraR and mutant rpsT P2 promoter
Components
  • (DNA (85-MER)) x 2
  • (DNA-directed RNA polymerase subunit ...Polymerase) x 4
  • Protein TraR
  • RNA polymerase sigma factor RpoD
Keywordstranscription/dna / TRANSCRIPTION / transcription-dna complex
Function / homology
Function and homology information


sigma factor antagonist complex / RNA polymerase complex / cytosolic DNA-directed RNA polymerase complex / regulation of DNA-templated transcription, initiation / sigma factor activity / transcription antitermination / ribonucleoside binding / DNA-directed 5'-3' RNA polymerase activity / DNA-directed RNA polymerase / DNA-templated transcription, initiation ...sigma factor antagonist complex / RNA polymerase complex / cytosolic DNA-directed RNA polymerase complex / regulation of DNA-templated transcription, initiation / sigma factor activity / transcription antitermination / ribonucleoside binding / DNA-directed 5'-3' RNA polymerase activity / DNA-directed RNA polymerase / DNA-templated transcription, initiation / protein-containing complex assembly / response to heat / transcription, DNA-templated / protein dimerization activity / response to antibiotic / magnesium ion binding / DNA binding / zinc ion binding / membrane / metal ion binding / cytosol / cytoplasm
Similarity search - Function
Zinc finger, C4 DksA/TraR-type / Zinc finger, DksA/TraR C4-type, bacteria / Zinc finger, DksA/TraR C4-type conserved site / Prokaryotic dksA C4-type zinc finger. / Prokaryotic dksA C4-type zinc finger profiles. / Zinc finger, DksA/TraR C4-type / Prokaryotic dksA/traR C4-type zinc finger / Sigma-70, non-essential region / RNA polymerase sigma factor 70, non-essential domain / Sigma-70 factor, region 1.1 superfamily ...Zinc finger, C4 DksA/TraR-type / Zinc finger, DksA/TraR C4-type, bacteria / Zinc finger, DksA/TraR C4-type conserved site / Prokaryotic dksA C4-type zinc finger. / Prokaryotic dksA C4-type zinc finger profiles. / Zinc finger, DksA/TraR C4-type / Prokaryotic dksA/traR C4-type zinc finger / Sigma-70, non-essential region / RNA polymerase sigma factor 70, non-essential domain / Sigma-70 factor, region 1.1 superfamily / RNA polymerase sigma factor 70, region 1.1 / Sigma-70 factor, region 1.1 / Sigma-70 factors family signature 1. / RNA polymerase sigma factor RpoD / RNA polymerase sigma factor RpoD, C-terminal / RNA polymerase subunit, RPB6/omega / Eukaryotic RPB6 RNA polymerase subunit / RNA polymerase sigma-70 region 1.2 / Sigma-70 factor, region 1.2 / Sigma-70 factors family signature 2. / RNA polymerase sigma-70 region 3 / DNA-directed RNA polymerase, insert domain / Sigma-70 region 3 / RNA polymerase sigma-70 / RNA polymerase, RBP11-like subunit / Sigma-70, region 4 / RNA polymerase sigma-70 region 4 / RNA Polymerase Alpha Subunit; Chain A, domain 2 / 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, beta subunit, external 1 domain superfamily / RNA polymerase, alpha subunit, C-terminal / Bacterial RNA polymerase, alpha chain C terminal domain / DNA-directed RNA polymerase, subunit beta-prime, bacterial type / DNA-directed RNA polymerase beta subunit, bacterial-type / DNA-directed RNA polymerase, beta subunit, external 1 domain / RNA polymerase beta subunit external 1 domain / DNA-directed RNA polymerase, alpha subunit / Gyrase A; domain 2 / RNA polymerase Rpb1, domain 3 superfamily / RNA polymerase Rpb1, clamp domain superfamily / DNA-directed RNA polymerase, subunit beta-prime / RNA polymerase Rpb6 / RNA polymerase, subunit omega/Rpo6/RPB6 / RNA polymerase Rpb6 / RPB6/omega subunit-like superfamily / RNA polymerase Rpb1, domain 1 / RNA polymerase Rpb1, funnel domain superfamily / RNA polymerase Rpb1, domain 2 / RNA polymerase Rpb1, domain 3 / RNA polymerase Rpb1, domain 3 / RNA polymerase Rpb1, domain 1 / RNA polymerase Rpb1, domain 5 / RNA polymerase, alpha subunit / RNA polymerase, N-terminal / RNA polymerase Rpb1, domain 4 / RNA polymerase Rpb1, domain 4 / RNA polymerase Rpb1, domain 5 / RNA polymerase I subunit A N-terminus / RNA polymerase Rpb2, domain 2 superfamily / RNA polymerase Rpb3/RpoA insert domain / DNA-directed RNA polymerase, insert domain / DNA-directed RNA polymerase, RpoA/D/Rpb3-type / RNA polymerase Rpb3/Rpb11 dimerisation domain / DNA-directed RNA polymerase, insert domain superfamily / RNA polymerases D / RNA polymerase, RBP11-like subunit / RNA polymerase Rpb2, domain 2 / RNA polymerase Rpb2, domain 2 / RNA polymerase beta subunit / RNA polymerase, beta subunit, protrusion / DNA-directed RNA polymerase, subunit 2, hybrid-binding domain / RNA polymerase Rpb2, domain 3 / RNA polymerase Rpb2, domain 7 / RNA polymerase, beta subunit, conserved site / RNA polymerase Rpb2, domain 7 / RNA polymerase Rpb2, domain 3 / RNA polymerase Rpb2, OB-fold / RNA polymerases beta chain signature. / DNA-directed RNA polymerase, subunit 2 / RNA polymerase Rpb2, domain 6 / DNA-directed RNA polymerase, subunit 2, hybrid-binding domain superfamily / 5' to 3' exonuclease, C-terminal subdomain / Beta Complex / DNA polymerase; domain 1 / Winged helix-like DNA-binding domain superfamily / Alpha-Beta Complex / 2-Layer Sandwich / Orthogonal Bundle / Mainly Beta / Mainly Alpha / Alpha Beta
Similarity search - Domain/homology
DNA (> 10) / DNA / RNA polymerase sigma factor RpoD / DNA-directed RNA polymerase subunit alpha / DNA-directed RNA polymerase subunit omega / DNA-directed RNA polymerase subunit omega / DNA-directed RNA polymerase subunit beta' / DNA-directed RNA polymerase subunit beta / DNA-directed RNA polymerase subunit beta / Protein TraR / RNA polymerase sigma factor RpoD
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3 Å
AuthorsChen, J. / Chiu, C.E. / Campbell, E.A. / Darst, S.A.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Human Genome Research Institute (NIH/NHGRI) United States
CitationJournal: Mol Cell / Year: 2020
Title: Stepwise Promoter Melting by Bacterial RNA Polymerase.
Authors: James Chen / Courtney Chiu / Saumya Gopalkrishnan / Albert Y Chen / Paul Dominic B Olinares / Ruth M Saecker / Jared T Winkelman / Michael F Maloney / Brian T Chait / Wilma Ross / Richard L ...Authors: James Chen / Courtney Chiu / Saumya Gopalkrishnan / Albert Y Chen / Paul Dominic B Olinares / Ruth M Saecker / Jared T Winkelman / Michael F Maloney / Brian T Chait / Wilma Ross / Richard L Gourse / Elizabeth A Campbell / Seth A Darst /
Abstract: Transcription initiation requires formation of the open promoter complex (RPo). To generate RPo, RNA polymerase (RNAP) unwinds the DNA duplex to form the transcription bubble and loads the DNA into ...Transcription initiation requires formation of the open promoter complex (RPo). To generate RPo, RNA polymerase (RNAP) unwinds the DNA duplex to form the transcription bubble and loads the DNA into the RNAP active site. RPo formation is a multi-step process with transient intermediates of unknown structure. We use single-particle cryoelectron microscopy to visualize seven intermediates containing Escherichia coli RNAP with the transcription factor TraR en route to forming RPo. The structures span the RPo formation pathway from initial recognition of the duplex promoter in a closed complex to the final RPo. The structures and supporting biochemical data define RNAP and promoter DNA conformational changes that delineate steps on the pathway, including previously undetected transient promoter-RNAP interactions that contribute to populating the intermediates but do not occur in RPo. Our work provides a structural basis for understanding RPo formation and its regulation, a major checkpoint in gene expression throughout evolution.
History
DepositionJul 13, 2019Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 25, 2020Provider: repository / Type: Initial release
Revision 1.1Apr 29, 2020Group: Database references / Category: citation / Item: _citation.journal_volume / _citation.page_first

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Structure visualization

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Assembly

Deposited unit
N: Protein TraR
O: DNA (85-MER)
P: DNA (85-MER)
M: DNA-directed RNA polymerase subunit alpha
G: DNA-directed RNA polymerase subunit alpha
H: DNA-directed RNA polymerase subunit alpha
I: DNA-directed RNA polymerase subunit beta
J: DNA-directed RNA polymerase subunit beta'
K: DNA-directed RNA polymerase subunit omega
L: RNA polymerase sigma factor RpoD
hetero molecules


Theoretical massNumber of molelcules
Total (without water)562,91418
Polymers560,16610
Non-polymers2,7488
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: mass spectrometry
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area56850 Å2
ΔGint-241 kcal/mol
Surface area166350 Å2

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Components

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Protein , 2 types, 2 molecules NL

#1: Protein Protein TraR / RNA polymerase transcription factor TraR


Mass: 8193.296 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: traR, ECOK12F083 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P41065
#8: Protein RNA polymerase sigma factor RpoD / Sigma-70


Mass: 70715.609 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: rpoD / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q0P6L9, UniProt: P00579*PLUS

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DNA chain , 2 types, 2 molecules OP

#2: DNA chain DNA (85-MER)


Mass: 26244.838 Da / Num. of mol.: 1 / Source method: obtained synthetically / Details: Non-template strand of rpsT P2 DNA promoter / Source: (synth.) Escherichia coli (E. coli)
#3: DNA chain DNA (85-MER)


Mass: 26191.744 Da / Num. of mol.: 1 / Source method: obtained synthetically / Details: Template strand of rpsT P2 DNA promoter / Source: (synth.) Escherichia coli (E. coli)

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

#4: 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.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: rpoA, pez, phs, sez, b3295, JW3257 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P0A7Z4, DNA-directed RNA polymerase
#5: 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 (E. coli) / Gene: rpoB, Z5560, ECs4910 / Production host: Escherichia coli BL21(DE3) (bacteria)
References: UniProt: P0A8V4, UniProt: P0A8V2*PLUS, DNA-directed RNA polymerase
#6: Protein DNA-directed RNA polymerase subunit beta' / Polymerase / RNAP subunit beta' / RNA polymerase subunit beta' / Transcriptase subunit beta'


Mass: 158073.609 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: rpoC, tabB, b3988, JW3951 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P0A8T7, DNA-directed RNA polymerase
#7: 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 (E. coli) / Gene: rpoZ, Z5075, ECs4524 / Production host: Escherichia coli BL21(DE3) (bacteria)
References: UniProt: P0A802, UniProt: P0A800*PLUS, DNA-directed RNA polymerase

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Non-polymers , 3 types, 8 molecules

#9: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: Zn
#10: Chemical
ChemComp-1N7 / CHAPSO / 2-hydroxy-N,N-dimethyl-3-sulfo-N-(3-{[(3beta,5beta,7beta,12beta)-3,7,12-trihydroxy-24-oxocholan-24-yl]amino}propyl)propan-1-aminium / CHAPS detergent


Mass: 631.884 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C32H59N2O8S / Comment: detergent*YM
#11: 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: Escherichia coli sigma70-holoenzyme bound to TraR and mutant rpsT P2 as TRPi1.5b
Type: COMPLEX / Entity ID: #1-#8 / Source: RECOMBINANT
Source (natural)Organism: Escherichia coli (E. coli)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 8
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 FIELDBright-field microscopy
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 93666 / Symmetry type: POINT

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