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- PDB-5nsr: Cryo-EM structure of RNA polymerase-sigma54 holo enzyme with prom... -

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Basic information

Entry
Database: PDB / ID: 5nsr
TitleCryo-EM structure of RNA polymerase-sigma54 holo enzyme with promoter DNA closed complex
Components
  • (DNA-directed RNA polymerase subunit ...Polymerase) x 4
  • Non-Template promoter DNA
  • RNA polymerase sigma-54 factor,RNA polymerase sigma-54 factor,RNA polymerase sigma-54 factor,RNA polymerase sigma-54 factor,RNA polymerase sigma-54 factor,RNA polymerase sigma-54 factor,RNA polymerase sigma-54 factor
  • Template DNA promoter
KeywordsTRANSCRIPTION / transcription initiation / RNA polymerase / sigma54
Function / homology
Function and homology information


nitrogen fixation / RNA polymerase complex / submerged biofilm formation / DNA-binding transcription activator activity / 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 ...nitrogen fixation / RNA polymerase complex / submerged biofilm formation / DNA-binding transcription activator activity / 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 / nucleotidyltransferase activity / 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 / magnesium ion binding / DNA binding / zinc ion binding / membrane / cytosol / cytoplasm
Similarity search - Function
Sigma-54 factors family signature 1. / Sigma-54 factors family profile. / RNA polymerase sigma factor 54, core-binding domain / RNA polymerase sigma factor 54, DNA-binding / RNA polymerase sigma-54 factor, core-binding domain superfamily / Sigma-54 factor, Activator interacting domain (AID) / Sigma-54, DNA binding domain / Sigma-54 factor, core binding domain / Sigma-54 factors family signature 2. / RNA polymerase sigma factor 54 ...Sigma-54 factors family signature 1. / Sigma-54 factors family profile. / RNA polymerase sigma factor 54, core-binding domain / RNA polymerase sigma factor 54, DNA-binding / RNA polymerase sigma-54 factor, core-binding domain superfamily / Sigma-54 factor, Activator interacting domain (AID) / Sigma-54, DNA binding domain / Sigma-54 factor, core binding domain / Sigma-54 factors family signature 2. / RNA polymerase sigma factor 54 / RNA polymerase subunit, RPB6/omega / Eukaryotic RPB6 RNA polymerase subunit / DNA-directed RNA polymerase, insert domain / RNA polymerase, RBP11-like subunit / RNA Polymerase Alpha Subunit; Chain A, domain 2 / Gyrase A; domain 2 / 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 / Beta Complex / 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 / Alpha-Beta Complex / 2-Layer Sandwich / Mainly Beta / Alpha Beta
Similarity search - Domain/homology
DNA / DNA (> 10) / RNA polymerase sigma-54 factor / RNA polymerase sigma-54 factor / 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)
Klebsiella pneumoniae (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.8 Å
AuthorsGlyde, R. / Ye, F.Z. / Darbari, V.C. / Zhang, N. / Buck, M. / Zhang, X.D.
Funding support United Kingdom, 2items
OrganizationGrant numberCountry
Biotechnology and Biological Sciences Research CouncilBB/N007816 United Kingdom
Wellcome TrustWT/098412 United Kingdom
CitationJournal: Mol Cell / Year: 2017
Title: Structures of RNA Polymerase Closed and Intermediate Complexes Reveal Mechanisms of DNA Opening and Transcription Initiation.
Authors: Robert Glyde / Fuzhou Ye / Vidya Chandran Darbari / Nan Zhang / Martin Buck / Xiaodong Zhang /
Abstract: Gene transcription is carried out by RNA polymerases (RNAPs). For transcription to occur, the closed promoter complex (RPc), where DNA is double stranded, must isomerize into an open promoter complex ...Gene transcription is carried out by RNA polymerases (RNAPs). For transcription to occur, the closed promoter complex (RPc), where DNA is double stranded, must isomerize into an open promoter complex (RPo), where the DNA is melted out into a transcription bubble and the single-stranded template DNA is delivered to the RNAP active site. Using a bacterial RNAP containing the alternative σ factor and cryoelectron microscopy, we determined structures of RPc and the activator-bound intermediate complex en route to RPo at 3.8 and 5.8 Å. Our structures show how RNAP-σ interacts with promoter DNA to initiate the DNA distortions required for transcription bubble formation, and how the activator interacts with RPc, leading to significant conformational changes in RNAP and σ that promote RPo formation. We propose that DNA melting is an active process initiated in RPc and that the RNAP conformations of intermediates are significantly different from that of RPc and RPo.
History
DepositionApr 26, 2017Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jun 28, 2017Provider: repository / Type: Initial release
Revision 1.1Jul 19, 2017Group: Database references / Category: citation
Item: _citation.country / _citation.journal_id_ASTM ..._citation.country / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.2Aug 2, 2017Group: Data collection / Category: em_image_scans / em_software / Item: _em_software.name
Revision 1.3Dec 11, 2019Group: Other / Category: atom_sites / cell
Item: _atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] ..._atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] / _atom_sites.fract_transf_matrix[3][3] / _cell.Z_PDB

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Structure visualization

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  • Deposited structure unit
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  • Superimposition on EM map
  • EMDB-3695
  • Imaged by UCSF Chimera
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Structure viewerMolecule:
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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
M: RNA polymerase sigma-54 factor,RNA polymerase sigma-54 factor,RNA polymerase sigma-54 factor,RNA polymerase sigma-54 factor,RNA polymerase sigma-54 factor,RNA polymerase sigma-54 factor,RNA polymerase sigma-54 factor
F: Non-Template promoter DNA
G: Template DNA promoter


Theoretical massNumber of molelcules
Total (without water)490,5578
Polymers490,5578
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration, native gel electrophoresis
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area31480 Å2
ΔGint-212 kcal/mol
Surface area180140 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 K-12 (bacteria) / Gene: rpoA, pez, phs, sez, b3295, JW3257 / Plasmid: pGEM / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): Gold pLysS AG / 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 K-12 (bacteria)
Gene: rpoB, groN, nitB, rif, ron, stl, stv, tabD, b3987, JW3950
Plasmid: pGEM / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): Gold pLysS AG / 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 K-12 (bacteria) / Gene: rpoC, tabB, b3988, JW3951 / Plasmid: pGEM / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): Gold pLysS AG / 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 K-12 (bacteria) / Gene: rpoZ, b3649, JW3624 / Plasmid: paCYCduet1-omega / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): Gold pLysS AG / References: UniProt: P0A800, DNA-directed RNA polymerase

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Protein , 1 types, 1 molecules M

#5: Protein RNA polymerase sigma-54 factor,RNA polymerase sigma-54 factor,RNA polymerase sigma-54 factor,RNA polymerase sigma-54 factor,RNA polymerase sigma-54 factor,RNA polymerase sigma-54 factor,RNA polymerase sigma-54 factor


Mass: 62158.797 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Klebsiella pneumoniae (bacteria) / Gene: rpoN, SAMEA3531867_02981, SM87_03359 / Plasmid: pET28b-sigma54 / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): Gold pLysS AG / References: UniProt: A0A0J4U551, UniProt: P06223*PLUS

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DNA chain , 2 types, 2 molecules FG

#6: DNA chain Non-Template promoter DNA


Mass: 19404.410 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)
#7: DNA chain Template DNA promoter


Mass: 19439.387 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Klebsiella pneumoniae (bacteria) / Gene: nifH / 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
1Cryo-EM structure of RNA polymerase-sigma54 holo enzyme with promoter DNA closed complexCOMPLEXall0RECOMBINANT
2Escherichia coli core RNA polymeraseCOMPLEX#1-#41RECOMBINANT
3Sigma-54 transcription initiation factorCOMPLEX#51RECOMBINANT
4Sigma-54 promoter DNACOMPLEX#6-#71RECOMBINANT
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Escherichia coli K-12 (bacteria)83333
23Klebsiella pneumoniae (bacteria)573
34Klebsiella pneumoniae (bacteria)573
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
12Escherichia coli BL21(DE3) (bacteria)469008
23Escherichia coli BL21(DE3) (bacteria)469008
34Escherichia coli BL21(DE3) (bacteria)469008
Buffer solutionpH: 8
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 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: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 1.4 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.10.1_2155: / Classification: refinement
EM software
IDNameVersionCategory
7UCSF Chimera1.9model fitting
8Coot0.8model fitting
13RELION1.43D reconstruction
14PHENIX1.10.1model refinement
15REFMACwithin package CCP4-7model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 80810 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00225818
ELECTRON MICROSCOPYf_angle_d0.61835500
ELECTRON MICROSCOPYf_dihedral_angle_d12.31419422
ELECTRON MICROSCOPYf_chiral_restr0.0434405
ELECTRON MICROSCOPYf_plane_restr0.0044513

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