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- PDB-6gh5: Cryo-EM structure of bacterial RNA polymerase-sigma54 holoenzyme ... -

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Basic information

Entry
Database: PDB / ID: 6gh5
TitleCryo-EM structure of bacterial RNA polymerase-sigma54 holoenzyme transcription open complex
Components
  • (DNA-directed RNA polymerase subunit ...Polymerase) x 4
  • (nifH promoter ...) x 2
  • 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
KeywordsTRANSCRIPTION / transcription initiation / DNA opening / transcription bubble / open complex
Function / homology
Function and homology information


RNA polymerase complex / nitrogen fixation / DNA-binding transcription activator activity / sigma factor activity / ribonucleoside binding / DNA-directed 5'-3' RNA polymerase activity / DNA-directed RNA polymerase / DNA-templated transcription, initiation / protein-containing complex assembly / protein dimerization activity ...RNA polymerase complex / nitrogen fixation / DNA-binding transcription activator activity / sigma factor activity / ribonucleoside binding / DNA-directed 5'-3' RNA polymerase activity / DNA-directed RNA polymerase / DNA-templated transcription, initiation / protein-containing complex assembly / protein dimerization activity / transcription, DNA-templated / response to antibiotic / magnesium ion binding / DNA binding / zinc ion binding / membrane / cytosol / cytoplasm
RNA polymerase, RBP11-like subunit / DNA-directed RNA polymerase, subunit 2 / 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, 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, RBP11-like subunit / DNA-directed RNA polymerase, subunit 2 / 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, 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 Rpb2, OB-fold / DNA-directed RNA polymerase, beta subunit, external 1 domain / RNA polymerase sigma factor 54, DNA-binding / RPB6/omega subunit-like superfamily / RNA polymerase beta subunit / DNA-directed RNA polymerase, insert domain superfamily / DNA-directed RNA polymerase, subunit 2, hybrid-binding domain superfamily / RNA polymerase Rpb2, domain 2 superfamily / RNA polymerase Rpb1, funnel domain superfamily / RNA polymerase sigma-54 factor, core-binding domain superfamily / RNA polymerase Rpb1, domain 3 superfamily / RNA polymerase Rpb2, domain 2 / RNA polymerase Rpb2, domain 7 / RNA polymerase Rpb2, domain 6 / Bacterial RNA polymerase, alpha chain C terminal domain / RNA polymerase Rpb2, domain 7 / RNA polymerase, beta subunit, conserved site / RNA polymerase Rpb3/RpoA insert domain / RNA polymerase beta subunit external 1 domain / Sigma-54 factor, core binding domain / Sigma-54, DNA binding domain / Sigma-54 factor, Activator interacting domain (AID) / RNA polymerase Rpb6 / RNA polymerase Rpb1, domain 4 / RNA polymerase Rpb1, domain 5 / RNA polymerase Rpb1, domain 1 / RNA polymerase Rpb1, domain 3 / DNA-directed RNA polymerase, subunit 2, hybrid-binding domain / RNA polymerase Rpb1, domain 2 / RNA polymerase Rpb2, domain 3 / RNA polymerase sigma factor 54 / RNA polymerase Rpb1, domain 4 / RNA polymerase Rpb1, domain 5 / RNA polymerase Rpb1, domain 1 / RNA polymerase Rpb1, domain 3 / RNA polymerase sigma factor 54, core-binding domain / RNA polymerase, N-terminal / RNA polymerase, subunit omega/K/RPB6 / DNA-directed RNA polymerase, omega subunit / RNA polymerase, alpha subunit / RNA polymerase Rpb3/Rpb11 dimerisation domain / 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 / 5' to 3' exonuclease, C-terminal subdomain / Beta Complex / DNA polymerase; domain 1 / Alpha-Beta Complex / 2-Layer Sandwich / Orthogonal Bundle / Mainly Beta / Mainly Alpha / Alpha Beta
DNA-directed RNA polymerase subunit beta' / 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
Biological speciesEscherichia coli (E. coli)
Klebsiella pneumoniae (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsGlyde, R. / Ye, F.Z. / Zhang, X.D.
Funding support United Kingdom, 1items
OrganizationGrant numberCountry
Biotechnology and Biological Sciences Research CouncilBB/N007816 United Kingdom
CitationJournal: Mol. Cell / Year: 2018
Title: Structures of Bacterial RNA Polymerase Complexes Reveal the Mechanism of DNA Loading and Transcription Initiation.
Authors: Robert Glyde / Fuzhou Ye / Milija Jovanovic / Ioly Kotta-Loizou / Martin Buck / Xiaodong Zhang /
Abstract: Gene transcription is carried out by multi-subunit RNA polymerases (RNAPs). Transcription initiation is a dynamic multi-step process that involves the opening of the double-stranded DNA to form a ...Gene transcription is carried out by multi-subunit RNA polymerases (RNAPs). Transcription initiation is a dynamic multi-step process that involves the opening of the double-stranded DNA to form a transcription bubble and delivery of the template strand deep into the RNAP for RNA synthesis. Applying cryoelectron microscopy to a unique transcription system using σ (σ), the major bacterial variant sigma factor, we capture a new intermediate state at 4.1 Å where promoter DNA is caught at the entrance of the RNAP cleft. Combining with new structures of the open promoter complex and an initial de novo transcribing complex at 3.4 and 3.7 Å, respectively, our studies reveal the dynamics of DNA loading and mechanism of transcription bubble stabilization that involves coordinated, large-scale conformational changes of the universally conserved features within RNAP and DNA. In addition, our studies reveal a novel mechanism of strand separation by σ.
Validation Report
SummaryFull reportAbout validation report
History
DepositionMay 4, 2018Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jul 4, 2018Provider: repository / Type: Initial release
Revision 1.1Oct 17, 2018Group: Data collection / Refinement description / Category: refine
Revision 1.2Dec 18, 2019Group: Other / Category: atom_sites
Item: _atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][1] ..._atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][1] / _atom_sites.fract_transf_matrix[2][2] / _atom_sites.fract_transf_matrix[3][2] / _atom_sites.fract_transf_matrix[3][3]

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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
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
F: nifH promoter template DNA
G: nifH promoter non-template DNA


Theoretical massNumber of molelcules
Total (without water)483,1698
Polymers483,1698
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration, native gel electrophoresis
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TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area42390 Å2
ΔGint-236 kcal/mol
Surface area173130 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 (strain K12) (bacteria)
Gene: rpoA, pez, phs, sez, b3295, JW3257
Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
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 (strain K12) (bacteria)
Gene: rpoB, groN, nitB, rif, ron, stl, stv, tabD, b3987, JW3950
Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
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 (strain K12) (bacteria)
Gene: rpoC, tabB, b3988, JW3951
Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
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 (strain K12) (bacteria)
Gene: rpoZ, b3649, JW3624
Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
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


Mass: 54723.082 Da / Num. of mol.: 1 / Mutation: R356A,R356A,R356A,R356A,R356A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Klebsiella pneumoniae (bacteria) / Gene: SM87_03359 / Production host: Escherichia coli K-12 (bacteria)
References: UniProt: A0A0J4U551, UniProt: P06223*PLUS, DNA-directed RNA polymerase

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NifH promoter ... , 2 types, 2 molecules FG

#6: DNA chain nifH promoter template DNA


Mass: 19404.410 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Klebsiella pneumoniae (bacteria)
#7: DNA chain nifH promoter non-template DNA


Mass: 19487.436 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Klebsiella pneumoniae (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 bacterial RNA polymerase-sigma54 holoenzyme transcription open complexCOMPLEX1, 2, 3, 4, 5, 6, 70MULTIPLE SOURCES
2DNA-directed RNA polymerasePolymeraseCOMPLEX1, 2, 3, 41RECOMBINANT
3RNA polymerase sigma-54 factorCOMPLEX51RECOMBINANT
4DNACOMPLEX6, 71RECOMBINANT
Molecular weightValue: 0.490 MDa / Experimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Escherichia coli K-12 (bacteria)83333
33Klebsiella pneumoniae (bacteria)573
44Klebsiella pneumoniae (bacteria)573
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
22Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)866768
33Escherichia coli K-12 (bacteria)83333
44synthetic construct (others)32630
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
110 mMTris hydrochlorideTris-HCLTris1
2150 mMsodium chlorideNaCLSodium chloride1
310 mMmagnesium chlorideMgCl21
SpecimenConc.: 0.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 95 % / 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: OTHER
Electron lensMode: BRIGHT FIELDBright-field microscopy
Image recordingElectron dose: 45 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

SoftwareName: REFMAC / Version: 5.8.0158 / Classification: refinement
EM software
IDNameVersionCategory
7UCSF Chimera1.9model fitting
8Coot8.03model fitting
10PHENIX1.10.1model refinement
11CCP4 package7model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 79678 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
RefinementResolution: 3.4→271.36 Å / Cor.coef. Fo:Fc: 0.918 / SU B: 10.109 / SU ML: 0.155 / ESU R: 0.16
Stereochemistry target values: MAXIMUM LIKELIHOOD WITH PHASES
RfactorNum. reflection% reflection
Rwork0.27208 --
Obs0.27208 1065335 100 %
Solvent computationSolvent model: PARAMETERS FOR MASK CACLULATION
Displacement parametersBiso mean: 250.91 Å2
Baniso -1Baniso -2Baniso -3
1-2.22 Å2-2.37 Å21.02 Å2
2---1.4 Å2-0.32 Å2
3----0.82 Å2
Refinement stepCycle: 1 / Total: 28366
Refine LS restraints
Refinement-IDTypeDev idealDev ideal targetNumber
ELECTRON MICROSCOPYr_bond_refined_d0.0340.01928943
ELECTRON MICROSCOPYr_bond_other_d
ELECTRON MICROSCOPYr_angle_refined_deg3.3821.91439752
ELECTRON MICROSCOPYr_angle_other_deg
ELECTRON MICROSCOPYr_dihedral_angle_1_deg8.46353628
ELECTRON MICROSCOPYr_dihedral_angle_2_deg2824.3931038
ELECTRON MICROSCOPYr_dihedral_angle_3_deg15.575154263
ELECTRON MICROSCOPYr_dihedral_angle_4_deg17.00715186
ELECTRON MICROSCOPYr_chiral_restr0.2260.24683
ELECTRON MICROSCOPYr_gen_planes_refined0.0190.02121070
ELECTRON MICROSCOPYr_gen_planes_other
ELECTRON MICROSCOPYr_nbd_refined
ELECTRON MICROSCOPYr_nbd_other
ELECTRON MICROSCOPYr_nbtor_refined
ELECTRON MICROSCOPYr_nbtor_other
ELECTRON MICROSCOPYr_xyhbond_nbd_refined
ELECTRON MICROSCOPYr_xyhbond_nbd_other
ELECTRON MICROSCOPYr_metal_ion_refined
ELECTRON MICROSCOPYr_metal_ion_other
ELECTRON MICROSCOPYr_symmetry_vdw_refined
ELECTRON MICROSCOPYr_symmetry_vdw_other
ELECTRON MICROSCOPYr_symmetry_hbond_refined
ELECTRON MICROSCOPYr_symmetry_hbond_other
ELECTRON MICROSCOPYr_symmetry_metal_ion_refined
ELECTRON MICROSCOPYr_symmetry_metal_ion_other
ELECTRON MICROSCOPYr_mcbond_it33.5923.64814557
ELECTRON MICROSCOPYr_mcbond_other
ELECTRON MICROSCOPYr_mcangle_it51.94435.36918170
ELECTRON MICROSCOPYr_mcangle_other
ELECTRON MICROSCOPYr_scbond_it49.629.30314386
ELECTRON MICROSCOPYr_scbond_other
ELECTRON MICROSCOPYr_scangle_it
ELECTRON MICROSCOPYr_scangle_other
ELECTRON MICROSCOPYr_long_range_B_refined94.895114698
ELECTRON MICROSCOPYr_long_range_B_other
ELECTRON MICROSCOPYr_rigid_bond_restr
ELECTRON MICROSCOPYr_sphericity_free
ELECTRON MICROSCOPYr_sphericity_bonded
LS refinement shellResolution: 3.4→3.488 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rwork0.701 79184 -
Rfree-0 -
Obs--100 %

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