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- PDB-5nss: Cryo-EM structure of RNA polymerase-sigma54 holoenzyme with promo... -

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Basic information

Entry
Database: PDB / ID: 5nss
TitleCryo-EM structure of RNA polymerase-sigma54 holoenzyme with promoter DNA and transcription activator PspF intermedate complex
Components
  • (DNA-directed RNA polymerase subunit ...Polymerase) x 4
  • Non-template promoter DNA
  • Psp operon transcriptional activator
  • 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,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,RNA polymerase sigma-54 factor,RNA polymerase sigma-54 factor,RNA polymerase sigma-54 factor RpoN,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,RNA polymerase sigma-54 factor
  • Template promoter DNA
KeywordsTRANSCRIPTION / transcription initiation / transcription intermediate complex / RNA polymerase / sigma54
Function / homology
Function and homology information


regulation of cellular response to stress / 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 ...regulation of cellular response to stress / 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 / phosphorelay signal transduction system / 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 / transcription regulator complex / sequence-specific DNA binding / protein dimerization activity / response to antibiotic / negative regulation of DNA-templated transcription / regulation of DNA-templated transcription / positive regulation of DNA-templated transcription / magnesium ion binding / ATP hydrolysis activity / DNA binding / zinc ion binding / ATP binding / membrane / identical protein binding / cytosol / cytoplasm
Similarity search - Function
Transcription activator PspF / 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. ...Transcription activator PspF / 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 interaction domain, conserved site / Sigma-54 interaction domain C-terminal part signature. / Sigma-54 interaction domain, ATP-binding site 2 / Sigma-54 interaction domain ATP-binding region B signature. / Sigma-54 interaction domain profile. / Sigma-54 interaction domain / RNA polymerase sigma factor 54 interaction domain / DNA binding HTH domain, Fis-type / Bacterial regulatory protein, Fis family / 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 / Homeobox-like domain superfamily / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
DNA / DNA (> 10) / RNA polymerase sigma-54 factor / 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 / Psp operon transcriptional activator
Similarity search - Component
Biological speciesEscherichia coli K-12 (bacteria)
Klebsiella pneumoniae (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 5.8 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
Revision 1.3Oct 24, 2018Group: Advisory / Data collection / Derived calculations
Category: pdbx_validate_close_contact / struct_conn / struct_conn_type
Revision 1.4Dec 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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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',DNA-directed RNA polymerase subunit beta',DNA-directed RNA polymerase subunit beta'
E: DNA-directed RNA polymerase subunit omega
F: Psp operon transcriptional activator
G: Psp operon transcriptional activator
J: Psp operon transcriptional activator
K: Psp operon transcriptional activator
L: Psp operon transcriptional activator
N: Psp operon transcriptional activator
H: Non-template promoter DNA
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,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,RNA polymerase sigma-54 factor,RNA polymerase sigma-54 factor,RNA polymerase sigma-54 factor RpoN,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,RNA polymerase sigma-54 factor
I: Template promoter DNA


Theoretical massNumber of molelcules
Total (without water)693,93914
Polymers693,93914
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration, native gel electrophoresis
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area29240 Å2
ΔGint-205 kcal/mol
Surface area187600 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: pET28b+ / 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',DNA-directed RNA polymerase subunit beta',DNA-directed RNA polymerase subunit beta' / Polymerase / RNAP subunit beta' / RNA polymerase subunit beta' / Transcriptase subunit beta'


Mass: 158941.062 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 / 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: pGEM / 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 , 2 types, 7 molecules FGJKLNM

#5: Protein
Psp operon transcriptional activator / Phage shock protein F


Mass: 33187.777 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli K-12 (bacteria) / Gene: pspF, ycjB, b1303, JW1296 / Plasmid: pGEM / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): Gold pLysS AG / References: UniProt: P37344
#7: 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,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,RNA polymerase sigma-54 factor,RNA polymerase sigma-54 factor,RNA polymerase sigma-54 factor RpoN,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,RNA polymerase sigma-54 factor


Mass: 62839.613 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Klebsiella pneumoniae (bacteria)
Gene: rpoN, KpST82_0597, SAMEA3531778_03572, SM87_03359, SAMEA4362693_02699, SM87_03359, SAMEA4362708_00719
Plasmid: pGEM / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): Gold pLysS AG
References: UniProt: A0A060VKE4, UniProt: A0A0J4U551, UniProt: A0A1W1PRD8, UniProt: P06223*PLUS

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DNA chain , 2 types, 2 molecules HI

#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.) Escherichia coli K-12 (bacteria) / Gene: rpoN / Plasmid: pET28b+ / Production host: Escherichia coli BL21(DE3) (bacteria)
#8: DNA chain Template promoter DNA


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) / Variant (production host): Gold pLysS AG

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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 holoenzyme with promoter DNA and transcription activator PspFCOMPLEXall0RECOMBINANT
2Escherichia coli core RNA polymeraseCOMPLEX#1-#51RECOMBINANT
3PspF transcriptional activatorCOMPLEX#61RECOMBINANT
4Sigma-54 transcription initiation factorCOMPLEX#81RECOMBINANT
5Sigma-54 promoter DNACOMPLEX#71RECOMBINANT
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Escherichia coli K-12 (bacteria)83333
23Escherichia coli K-12 (bacteria)83333
34Klebsiella pneumoniae (bacteria)573
45Klebsiella pneumoniae (bacteria)573
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
12Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)866768
23Escherichia coli BL21(DE3) (bacteria)469008
34Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)866768
45Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)866768
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: 1.25 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 softwareName: RELION / Version: 1.4 / Category: 3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 5.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 79355 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00232721
ELECTRON MICROSCOPYf_angle_d0.55745228
ELECTRON MICROSCOPYf_dihedral_angle_d10.70122421
ELECTRON MICROSCOPYf_chiral_restr0.0435783
ELECTRON MICROSCOPYf_plane_restr0.0035935

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