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- PDB-6eec: Mycobacterium tuberculosis RNAP promoter unwinding intermediate c... -

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Basic information

Entry
Database: PDB / ID: 6eec
TitleMycobacterium tuberculosis RNAP promoter unwinding intermediate complex with RbpA/CarD and AP3 promoter captured by Corallopyronin
Components
  • (DNA-directed RNA polymerase subunit ...Polymerase) x 4
  • (RNA polymerase-binding ...) x 2
  • DNA (63-MER)
  • DNA (65-MER)
  • RNA polymerase sigma factor SigA
KeywordsTRANSCRIPTION/DNA / initiation / transcription bubble / closed clamp / open promoter complex / TRANSCRIPTION / TRANSCRIPTION-DNA complex
Function / homology
Function and homology information


bacterial-type RNA polymerase holo enzyme binding / regulation of growth rate / response to water / bacterial-type RNA polymerase core enzyme binding / transcription initiation from bacterial-type RNA polymerase promoter / sigma factor activity / stringent response / ribonucleoside binding / DNA-directed 5'-3' RNA polymerase activity / DNA-directed RNA polymerase ...bacterial-type RNA polymerase holo enzyme binding / regulation of growth rate / response to water / bacterial-type RNA polymerase core enzyme binding / transcription initiation from bacterial-type RNA polymerase promoter / sigma factor activity / stringent response / ribonucleoside binding / DNA-directed 5'-3' RNA polymerase activity / DNA-directed RNA polymerase / cell wall / pathogenesis / protein dimerization activity / transcription, DNA-templated / response to antibiotic / DNA-binding transcription factor activity / positive regulation of transcription, DNA-templated / DNA binding / plasma membrane / cytosol / cytoplasm
RNA polymerase Rpb2, OB-fold / RNA polymerase sigma factor, region 3/4-like / RNA polymerase sigma factor RpoD / RNA polymerase-binding protein RbpA / DNA-directed RNA polymerase, beta subunit, external 1 domain / DNA-directed RNA polymerase, subunit 2 / RNA polymerase sigma-70 like domain / RNA polymerase sigma factor, region 2 / RNA polymerase sigma factor RpoD, C-terminal / RPB6/omega subunit-like superfamily ...RNA polymerase Rpb2, OB-fold / RNA polymerase sigma factor, region 3/4-like / RNA polymerase sigma factor RpoD / RNA polymerase-binding protein RbpA / DNA-directed RNA polymerase, beta subunit, external 1 domain / DNA-directed RNA polymerase, subunit 2 / RNA polymerase sigma-70 like domain / RNA polymerase sigma factor, region 2 / RNA polymerase sigma factor RpoD, C-terminal / RPB6/omega subunit-like superfamily / DNA-directed RNA polymerase, subunit beta-prime / RNA polymerase subunit, RPB6/omega / DNA-directed RNA polymerase, alpha subunit / DNA-directed RNA polymerase, RpoA/D/Rpb3-type / DNA-directed RNA polymerase, insert domain / RNA polymerase, alpha subunit, C-terminal / DNA-directed RNA polymerase beta subunit, bacterial-type / CarD-like/TRCF domain superfamily / Winged helix-like DNA-binding domain superfamily / RNA polymerase Rpb2, domain 2 / RNA polymerase Rpb3/RpoA insert domain / Sigma-70, region 4 / Sigma-70 region 2 / Sigma-70 region 3 / Bacterial RNA polymerase, alpha chain C terminal domain / CarD-like/TRCF domain / RNA polymerase Rpb3/Rpb11 dimerisation domain / RNA polymerase Rpb6 / RNA polymerase Rpb1, domain 2 / RNA polymerase, RBP11-like subunit / RNA polymerase Rpb2, domain 6 / Sigma-70 factor, region 1.2 / RbpA superfamily / RNA polymerase Rpb1, funnel domain superfamily / RNA polymerase Rpb2, domain 2 superfamily / DNA-directed RNA polymerase, subunit 2, hybrid-binding domain superfamily / DNA-directed RNA polymerase, insert domain superfamily / RNA polymerase sigma-70 region 1.2 / RNA polymerase Rpb2, domain 3 / RNA polymerase, beta subunit, protrusion / CarD-like/TRCF domain / RNA polymerases beta chain signature. / Sigma-70 factors family signature 2. / Sigma-70 factors family signature 1. / RNA polymerase-binding protein / RNA polymerase beta subunit external 1 domain / RNA polymerase Rpb1, domain 4 / RNA polymerase Rpb1, domain 5 / RNA polymerase Rpb1, domain 1 / RNA polymerase Rpb1, domain 3 / RNA polymerase Rpb2, domain 3 / RNA polymerase Rpb2, domain 2 / RNA polymerase, alpha subunit / RNA polymerase sigma-70 / RNA polymerase beta subunit / DNA-directed RNA polymerase, omega subunit / RNA polymerase, subunit omega/K/RPB6 / RNA polymerase Rpb2, domain 7 / RNA polymerase sigma-70 region 4 / RNA polymerase sigma-70 region 2 / RNA polymerase sigma-70 region 3 / RNA polymerase, beta subunit, conserved site / DNA-directed RNA polymerase, subunit 2, hybrid-binding domain / RNA polymerase Rpb1, domain 4 / RNA polymerase Rpb1, domain 5 / RNA polymerase Rpb1, domain 3 / RNA polymerase Rpb1, domain 1 / RNA polymerase, N-terminal / RNA polymerase Rpb2, domain 7
DNA-directed RNA polymerase subunit beta / RNA polymerase-binding transcription factor CarD / RNA polymerase-binding transcription factor CarD / RNA polymerase-binding protein RbpA / RNA polymerase-binding protein RbpA / DNA-directed RNA polymerase subunit alpha / DNA-directed RNA polymerase subunit beta' / DNA-directed RNA polymerase subunit beta' / DNA-directed RNA polymerase subunit omega / RNA polymerase sigma factor SigA ...DNA-directed RNA polymerase subunit beta / RNA polymerase-binding transcription factor CarD / RNA polymerase-binding transcription factor CarD / RNA polymerase-binding protein RbpA / RNA polymerase-binding protein RbpA / DNA-directed RNA polymerase subunit alpha / DNA-directed RNA polymerase subunit beta' / DNA-directed RNA polymerase subunit beta' / DNA-directed RNA polymerase subunit omega / RNA polymerase sigma factor SigA / RNA polymerase sigma factor SigA / DNA-directed RNA polymerase subunit alpha / DNA-directed RNA polymerase subunit omega / DNA-directed RNA polymerase subunit beta
Specimen sourceMycobacterium tuberculosis (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.55 Å
AuthorsDarst, S.A. / Campbell, E.A. / Boyaci Selcuk, H. / Chen, J.
CitationJournal: Nature / Year: 2019
Title: Structures of an RNA polymerase promoter melting intermediate elucidate DNA unwinding.
Authors: Hande Boyaci / James Chen / Rolf Jansen / Seth A Darst / Elizabeth A Campbell /
Abstract: A key regulated step of transcription is promoter melting by RNA polymerase (RNAP) to form the open promoter complex. To generate the open complex, the conserved catalytic core of the RNAP combines ...A key regulated step of transcription is promoter melting by RNA polymerase (RNAP) to form the open promoter complex. To generate the open complex, the conserved catalytic core of the RNAP combines with initiation factors to locate promoter DNA, unwind 12-14 base pairs of the DNA duplex and load the template-strand DNA into the RNAP active site. Formation of the open complex is a multi-step process during which transient intermediates of unknown structure are formed. Here we present cryo-electron microscopy structures of bacterial RNAP-promoter DNA complexes, including structures of partially melted intermediates. The structures show that late steps of promoter melting occur within the RNAP cleft, delineate key roles for fork-loop 2 and switch 2-universal structural features of RNAP-in restricting access of DNA to the RNAP active site, and explain why clamp opening is required to allow entry of single-stranded template DNA into the active site. The key roles of fork-loop 2 and switch 2 suggest a common mechanism for late steps in promoter DNA opening to enable gene expression across all domains of life.
Validation Report
SummaryFull reportAbout validation report
DateDeposition: Aug 13, 2018 / Release: Nov 21, 2018
RevisionDateData content typeGroupCategoryItemProviderType
1.0Nov 21, 2018Structure modelrepositoryInitial release
1.1Jan 23, 2019Structure modelData collection / Database referencescitation / citation_author_citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year
1.2Jan 30, 2019Structure modelData collection / Database referencescitation_citation.journal_volume / _citation.page_first / _citation.page_last

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Structure visualization

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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 SigA
J: RNA polymerase-binding protein RbpA
O: DNA (65-MER)
P: DNA (63-MER)
M: RNA polymerase-binding transcription factor CarD
hetero molecules


Theoretical massNumber of molelcules
Total (without water)510,84614
Polyers510,16410
Non-polymers6834
Water0
1


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/peptide DNA-directed RNA polymerase subunit alpha / Polymerase / RNAP subunit alpha / RNA polymerase subunit alpha / Transcriptase subunit alpha


Mass: 37745.328 Da / Num. of mol.: 2 / Source: (gene. exp.) Mycobacterium tuberculosis (bacteria) / Gene: rpoA / Production host: Escherichia coli (E. coli)
References: UniProt: A5U8D3, UniProt: P9WGZ1*PLUS, DNA-directed RNA polymerase
#2: Protein/peptide DNA-directed RNA polymerase subunit beta / Polymerase / RNAP subunit beta / RNA polymerase subunit beta / Transcriptase subunit beta


Mass: 130070.797 Da / Num. of mol.: 1 / Source: (gene. exp.) Mycobacterium tuberculosis (bacteria) / Gene: rpoB, SAMEA2682864_01701 / Production host: Escherichia coli (E. coli)
References: UniProt: V9Z879, UniProt: P9WGY9*PLUS, DNA-directed RNA polymerase
#3: Protein/peptide DNA-directed RNA polymerase subunit beta' / Polymerase / RNAP subunit beta' / RNA polymerase subunit beta' / Transcriptase subunit beta'


Mass: 148202.219 Da / Num. of mol.: 1 / Source: (gene. exp.) Mycobacterium tuberculosis (bacteria) / Gene: rpoC / Production host: Escherichia coli (E. coli)
References: UniProt: A5U053, UniProt: P9WGY7*PLUS, DNA-directed RNA polymerase
#4: Protein/peptide DNA-directed RNA polymerase subunit omega / Polymerase / RNAP omega subunit / RNA polymerase omega subunit / Transcriptase subunit omega


Mass: 11776.996 Da / Num. of mol.: 1 / Source: (gene. exp.) Mycobacterium tuberculosis (bacteria)
Gene: rpoZ, ERS007672_03979, ERS007703_04032, ERS007720_04749, ERS027652_00548, ERS027654_02543, ERS027656_03959, ERS124361_02246
Production host: Escherichia coli (E. coli)
References: UniProt: A0A0T9N9K3, UniProt: P9WGY5*PLUS, DNA-directed RNA polymerase

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Protein/peptide , 1 types, 1 molecules F

#5: Protein/peptide RNA polymerase sigma factor SigA / Sigma-A


Mass: 58169.477 Da / Num. of mol.: 1 / Source: (gene. exp.) Mycobacterium tuberculosis (bacteria) / Gene: sigA, mysA, rpoD, rpoV / Production host: Escherichia coli (E. coli) / References: UniProt: P9WGI0, UniProt: P9WGI1*PLUS

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RNA polymerase-binding ... , 2 types, 2 molecules JM

#6: Protein/peptide RNA polymerase-binding protein RbpA


Mass: 12993.695 Da / Num. of mol.: 1 / Source: (gene. exp.) Mycobacterium tuberculosis (bacteria) / Gene: rbpA / Production host: Escherichia coli (E. coli) / References: UniProt: P9WHJ4, UniProt: P9WHJ5*PLUS
#9: Protein/peptide RNA polymerase-binding transcription factor CarD


Mass: 17933.361 Da / Num. of mol.: 1 / Source: (gene. exp.) Mycobacterium tuberculosis (bacteria) / Gene: carD / Production host: Escherichia coli (E. coli) / References: UniProt: P9WJG2, UniProt: P9WJG3*PLUS

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DNA chain , 2 types, 2 molecules OP

#7: DNA chain DNA (65-MER)


Mass: 27907.809 Da / Num. of mol.: 1 / Source: (synth.) Mycobacterium tuberculosis (bacteria)
#8: DNA chain DNA (63-MER)


Mass: 27618.625 Da / Num. of mol.: 1 / Source: (synth.) Mycobacterium tuberculosis (bacteria)

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Non-polymers , 3 types, 4 molecules

#10: Chemical ChemComp-C0L / methyl [(1E,5R)-5-{(3E)-3-[(2E,4E,8R,9E,12E)-1,8-dihydroxy-2,5,9-trimethyltetradeca-2,4,9,12-tetraen-1-ylidene]-2,4-dioxo-3,4-dihydro-2H-pyran-6-yl}hex-1-en-1-yl]carbamate / Corallopyronin A


Mass: 527.649 Da / Num. of mol.: 1 / Formula: C30H41NO7
#11: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 2 / Formula: Zn / Zinc
#12: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Formula: Mg / Magnesium

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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: Mycobacterium tuberculosis RNAP open promoter complex / Type: COMPLEX / Entity ID: 1,2,3,4,5,6,7,8,9 / Source: RECOMBINANT
Source (natural)Organism: Mycobacterium tuberculosis (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 8
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: unspecified
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: 69.9 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.13_2998: / Classification: refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.55 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 246409 / Symmetry type: POINT
Refine LS restraints

Refinement-ID: ELECTRON MICROSCOPY

TypeDev idealNumber
f_bond_d0.00930696
f_angle_d0.86142154
f_dihedral_angle_d13.18918275
f_chiral_restr0.0484789
f_plane_restr0.0055082

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