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- EMDB-9037: Mycobacterium tuberculosis RNAP open promoter complex with RbpA/C... -

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Basic information

Entry
Database: EMDB / ID: EMD-9037
TitleMycobacterium tuberculosis RNAP open promoter complex with RbpA/CarD and AP3 promoter
Map data
SampleMycobacterium tuberculosis RNAP open promoter complex
  • (DNA-directed RNA polymerase subunit ...Polymerase) x 4
  • RNA polymerase sigma factor SigA
  • (RNA polymerase-binding ...) x 2
  • (nucleic-acidNucleic acid) x 2
  • (ligand) x 2
Function / homology
Function and homology information


bacterial-type RNA polymerase holo enzyme binding / response to water / bacterial-type RNA polymerase core enzyme binding / regulation of growth rate / 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 / response to water / bacterial-type RNA polymerase core enzyme binding / regulation of growth rate / 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 / protein dimerization activity / transcription, DNA-templated / response to antibiotic / DNA-binding transcription factor activity / pathogenesis / positive regulation of transcription, DNA-templated / magnesium ion binding / DNA binding / zinc ion binding / plasma membrane / cytosol / cytoplasm
RNA polymerase sigma-70 region 1.2 / RNA polymerase sigma factor RpoD / RNA polymerase Rpb1, domain 5 / RNA polymerase Rpb1, domain 4 / DNA-directed RNA polymerase, subunit 2, hybrid-binding domain / RNA polymerase, beta subunit, conserved site / RNA polymerase sigma-70 region 3 / RNA polymerase sigma-70 region 2 / RNA polymerase sigma-70 region 4 / RNA polymerase Rpb2, domain 7 ...RNA polymerase sigma-70 region 1.2 / RNA polymerase sigma factor RpoD / RNA polymerase Rpb1, domain 5 / RNA polymerase Rpb1, domain 4 / DNA-directed RNA polymerase, subunit 2, hybrid-binding domain / RNA polymerase, beta subunit, conserved site / RNA polymerase sigma-70 region 3 / RNA polymerase sigma-70 region 2 / RNA polymerase sigma-70 region 4 / RNA polymerase Rpb2, domain 7 / RNA polymerase Rpb2, domain 2 / RNA polymerase, beta subunit, protrusion / RNA polymerase Rpb2, domain 3 / DNA-directed RNA polymerase beta subunit, bacterial-type / RNA polymerase Rpb1, domain 3 / RNA polymerase, alpha subunit, C-terminal / DNA-directed RNA polymerase, insert domain / DNA-directed RNA polymerase, RpoA/D/Rpb3-type / DNA-directed RNA polymerase, alpha subunit / RNA polymerase subunit, RPB6/omega / DNA-directed RNA polymerase, subunit beta-prime / RNA polymerase sigma factor RpoD, C-terminal / RNA polymerase sigma factor, region 3/4-like / RNA polymerase sigma factor, region 2 / RNA polymerase sigma-70 like domain / RNA polymerase Rpb2, OB-fold / DNA-directed RNA polymerase, subunit 2 / RNA polymerase Rpb1, domain 1 / RNA polymerase, N-terminal / RNA polymerase-binding protein RbpA / RNA polymerase, subunit omega/K/RPB6 / CarD-like, C-terminal domain / DNA-directed RNA polymerase, beta subunit, external 1 domain superfamily / RNA polymerase Rpb1, domain 3 superfamily / 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, RBP11-like subunit / DNA-directed RNA polymerase, beta subunit, external 1 domain / CarD-like/TRCF domain superfamily / DNA-directed RNA polymerase, omega subunit / CarD-like/TRCF domain / RNA polymerase sigma-70 / RNA polymerase, alpha subunit / RPB6/omega subunit-like superfamily / Winged helix-like DNA-binding domain superfamily
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 omega / 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 omega / 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 beta' / DNA-directed RNA polymerase subunit beta
Biological speciesMycobacterium tuberculosis (bacteria)
Methodsingle particle reconstruction / cryo EM
AuthorsDarst SA / Campbell EA / 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 ReportPDB-ID: 6edt

SummaryFull reportAbout validation report
History
Header (metadata) releaseFeb 28, 2018-
DepositionAug 10, 2018-
Map releaseNov 21, 2018-
UpdateDec 18, 2019-
Current statusDec 18, 2019Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.3
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.3
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: : PDB-6edt
  • Surface level: 0.3
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_9037.map.gz / Format: CCP4 / Size: 59.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.3 Å/pix.
x 250 pix.
= 325. Å
1.3 Å/pix.
x 250 pix.
= 325. Å
1.3 Å/pix.
x 250 pix.
= 325. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel size
XYZ
EMDB info.1.071.071.07
CCP4 map header1.31.31.3
EM Navigator Movie #11.31.31.3
Density
Contour LevelBy AUTHOR: 0.25 / Movie #1: 0.3
Minimum - Maximum-0.7299977 - 1.9311254
Average (Standard dev.)0.006558573 (±0.07501595)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions250250250
Spacing250250250
CellA=B=C: 267.5 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.31.31.3
M x/y/z250250250
origin x/y/z0.0000.0000.000
length x/y/z325.000325.000325.000
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ240240240
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS250250250
D min/max/mean-0.7301.9310.007

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Supplemental data

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Sample components

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Entire Mycobacterium tuberculosis RNAP open promoter complex

EntireName: Mycobacterium tuberculosis RNAP open promoter complex / Number of components: 12

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Component #1: protein, Mycobacterium tuberculosis RNAP open promoter complex

ProteinName: Mycobacterium tuberculosis RNAP open promoter complex / Recombinant expression: No
SourceSpecies: Mycobacterium tuberculosis (bacteria)
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #2: protein, DNA-directed RNA polymerase subunit alpha

ProteinName: DNA-directed RNA polymerase subunit alphaPolymerase / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 37.745328 kDa
SourceSpecies: Mycobacterium tuberculosis (bacteria)
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #3: protein, DNA-directed RNA polymerase subunit beta

ProteinName: DNA-directed RNA polymerase subunit betaPolymerase / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 125.390875 kDa
SourceSpecies: Mycobacterium tuberculosis (bacteria)
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #4: protein, DNA-directed RNA polymerase subunit beta'

ProteinName: DNA-directed RNA polymerase subunit beta'Polymerase / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 152.882109 kDa
SourceSpecies: Mycobacterium tuberculosis (bacteria)
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #5: protein, DNA-directed RNA polymerase subunit omega

ProteinName: DNA-directed RNA polymerase subunit omegaPolymerase / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 11.776996 kDa
SourceSpecies: Mycobacterium tuberculosis (bacteria)
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #6: protein, RNA polymerase sigma factor SigA

ProteinName: RNA polymerase sigma factor SigA / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 58.169477 kDa
SourceSpecies: Mycobacterium tuberculosis (bacteria)
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #7: protein, RNA polymerase-binding protein RbpA

ProteinName: RNA polymerase-binding protein RbpA / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 12.993695 kDa
SourceSpecies: Mycobacterium tuberculosis (bacteria)
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #8: nucleic-acid, DNA (65-MER)

nucleic acidName: DNA (65-MER) / Class: DNA / Structure: OTHER / Synthetic: No
Sequence: (DG)(DG)(DC)(DT)(DA)(DT)(DG)(DG)(DA)(DT) (DG)(DA)(DC)(DC)(DG)(DA)(DA)(DC)(DC)(DT) (DG)(DG)(DT)(DC)(DT)(DT)(DG)(DA)(DC)(DT) (DC)(DC)(DA)(DT)(DT)(DG)(DC)(DC)(DG)(DG) (DA)(DT)(DT)(DT)(DG)(DT) ...Sequence:
(DG)(DG)(DC)(DT)(DA)(DT)(DG)(DG)(DA)(DT) (DG)(DA)(DC)(DC)(DG)(DA)(DA)(DC)(DC)(DT) (DG)(DG)(DT)(DC)(DT)(DT)(DG)(DA)(DC)(DT) (DC)(DC)(DA)(DT)(DT)(DG)(DC)(DC)(DG)(DG) (DA)(DT)(DT)(DT)(DG)(DT)(DA)(DT)(DT)(DA) (DG)(DA)(DC)(DT)(DG)(DG)(DC)(DA)(DG)(DG) (DG)(DT)(DT)(DG)(DC)(DC)(DC)(DC)(DG)(DA) (DA)(DG)(DC)(DG)(DG)(DG)(DC)(DG)(DG)(DA) (DA)(DA)(DC)(DA)(DA)(DG)(DC)(DA)(DC)(DG)
MassTheoretical: 27.907809 kDa
SourceSpecies: Mycobacterium tuberculosis (bacteria)

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Component #9: nucleic-acid, DNA (65-MER)

nucleic acidName: DNA (65-MER) / Class: DNA / Structure: OTHER / Synthetic: No
Sequence: (DC)(DG)(DT)(DG)(DC)(DT)(DT)(DG)(DT)(DT) (DT)(DC)(DC)(DG)(DC)(DC)(DC)(DG)(DC)(DT) (DT)(DC)(DG)(DG)(DG)(DG)(DC)(DA)(DA)(DC) (DC)(DC)(DT)(DG)(DC)(DC)(DA)(DG)(DT)(DC) (DT)(DA)(DA)(DT)(DA)(DC) ...Sequence:
(DC)(DG)(DT)(DG)(DC)(DT)(DT)(DG)(DT)(DT) (DT)(DC)(DC)(DG)(DC)(DC)(DC)(DG)(DC)(DT) (DT)(DC)(DG)(DG)(DG)(DG)(DC)(DA)(DA)(DC) (DC)(DC)(DT)(DG)(DC)(DC)(DA)(DG)(DT)(DC) (DT)(DA)(DA)(DT)(DA)(DC)(DA)(DA)(DA)(DT) (DC)(DC)(DG)(DG)(DC)(DA)(DA)(DT)(DG)(DG) (DA)(DG)(DT)(DC)(DA)(DA)(DG)(DA)(DC)(DC) (DA)(DG)(DG)(DT)(DT)(DC)(DG)(DG)(DT)(DC) (DA)(DT)(DC)(DC)(DA)(DT)(DA)(DG)(DC)(DC)
MassTheoretical: 27.618625 kDa
SourceSpecies: Mycobacterium tuberculosis (bacteria)

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Component #10: protein, RNA polymerase-binding transcription factor CarD

ProteinName: RNA polymerase-binding transcription factor CarD / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 17.933361 kDa
SourceSpecies: Mycobacterium tuberculosis (bacteria)
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #11: ligand, ZINC ION

LigandName: ZINC ION / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 6.540905 MDa

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Component #12: ligand, MAGNESIUM ION

LigandName: MAGNESIUM ION / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 2.430505 MDa

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Experimental details

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Sample preparation

SpecimenSpecimen state: Particle / Method: cryo EM
Sample solutionpH: 8
Support filmunspecified
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
ImagingMicroscope: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 69.9 e/Å2 / Illumination mode: FLOOD BEAM
LensImaging mode: BRIGHT FIELD
Specimen HolderModel: OTHER
CameraDetector: GATAN K2 SUMMIT (4k x 4k)

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Image processing

ProcessingMethod: single particle reconstruction / Number of projections: 211381

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Atomic model buiding

Output model

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