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- PDB-6alh: CryoEM structure of E.coli RNA polymerase elongation complex -

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Basic information

Entry
Database: PDB / ID: 6alh
TitleCryoEM structure of E.coli RNA polymerase elongation complex
Components
  • (DNA-directed RNA polymerase subunit ...Polymerase) x 4
  • DNA (29-MER)
  • DNA (5'-D(*GP*GP*GP*CP*TP*AP*AP*TP*GP*AP*CP*GP*GP*CP*GP*AP*AP*TP*AP*CP*CP*C)-3')
  • RNA (5'-R(P*CP*GP*GP*AP*GP*AP*GP*GP*UP*A)-3')
Keywordstranscription/dna/rna / DNA-dependent RNA polymerase / TRANSCRIPTION / transcription-dna-rna complex
Function / homology
Function and homology information


RNA polymerase complex / ribonucleoside binding / DNA-directed 5'-3' RNA polymerase activity / DNA-directed RNA polymerase / protein-containing complex assembly / protein dimerization activity / transcription, DNA-templated / response to antibiotic / magnesium ion binding / DNA binding ...RNA polymerase complex / ribonucleoside binding / DNA-directed 5'-3' RNA polymerase activity / DNA-directed RNA polymerase / 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 Rpb2, domain 7 / RNA polymerase Rpb1, domain 3 / 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 / RNA polymerase Rpb2, domain 3 / RNA polymerase, beta subunit, protrusion / RNA polymerase Rpb2, domain 2 ...RNA polymerase Rpb2, domain 7 / RNA polymerase Rpb1, domain 3 / 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 / RNA polymerase Rpb2, domain 3 / RNA polymerase, beta subunit, protrusion / RNA polymerase Rpb2, domain 2 / RNA polymerase Rpb2, OB-fold / 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 1 / RNA polymerase, N-terminal / DNA-directed RNA polymerase, subunit beta-prime / RNA polymerase, RBP11-like subunit / RNA polymerase Rpb1, domain 3 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 subunit, RPB6/omega / RNA polymerase, subunit omega/K/RPB6 / RPB6/omega subunit-like superfamily / DNA-directed RNA polymerase, beta subunit, external 1 domain / DNA-directed RNA polymerase, subunit 2 / RNA polymerase, alpha subunit / DNA-directed RNA polymerase, omega subunit
DNA-directed RNA polymerase subunit alpha / DNA-directed RNA polymerase subunit omega / DNA-directed RNA polymerase subunit beta' / DNA-directed RNA polymerase subunit beta
Biological speciesEscherichia coli (E. coli)
Enterobacteria phage T7 (bacteriophage)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.4 Å
AuthorsKang, J.Y. / Darst, S.A.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35 GM118130 United States
CitationJournal: Elife / Year: 2017
Title: Structural basis of transcription arrest by coliphage HK022 Nun in an RNA polymerase elongation complex.
Authors: Jin Young Kang / Paul Dominic B Olinares / James Chen / Elizabeth A Campbell / Arkady Mustaev / Brian T Chait / Max E Gottesman / Seth A Darst /
Abstract: Coliphage HK022 Nun blocks superinfection by coliphage λ by stalling RNA polymerase (RNAP) translocation specifically on λ DNA. To provide a structural framework to understand how Nun blocks RNAP ...Coliphage HK022 Nun blocks superinfection by coliphage λ by stalling RNA polymerase (RNAP) translocation specifically on λ DNA. To provide a structural framework to understand how Nun blocks RNAP translocation, we determined structures of RNAP ternary elongation complexes (TECs) with and without Nun by single-particle cryo-electron microscopy. Nun fits tightly into the TEC by taking advantage of gaps between the RNAP and the nucleic acids. The C-terminal segment of Nun interacts with the RNAP β and β' subunits inside the RNAP active site cleft as well as with nearly every element of the nucleic acid scaffold, essentially crosslinking the RNAP and the nucleic acids to prevent translocation, a mechanism supported by the effects of Nun amino acid substitutions. The nature of Nun interactions inside the RNAP active site cleft suggests that RNAP clamp opening is required for Nun to establish its interactions, explaining why Nun acts on paused TECs.
Validation Report
SummaryFull reportAbout validation report
History
DepositionAug 7, 2017Deposition site: RCSB / Processing site: RCSB
SupersessionAug 16, 2017ID: 5UPC
Revision 1.0Aug 16, 2017Provider: repository / Type: Initial release
Revision 1.1Sep 20, 2017Group: Author supporting evidence / Data collection / Category: em_software / pdbx_audit_support
Item: _em_software.name / _pdbx_audit_support.funding_organization
Revision 1.2Nov 8, 2017Group: Derived calculations / Category: pdbx_struct_assembly
Item: _pdbx_struct_assembly.details / _pdbx_struct_assembly.method_details
Revision 1.3Feb 28, 2018Group: Database references / Other / Category: cell / citation
Item: _cell.Z_PDB / _cell.length_a ..._cell.Z_PDB / _cell.length_a / _cell.length_b / _cell.length_c / _citation.title
Revision 1.4Mar 28, 2018Group: Data collection / Database references / Category: citation / Item: _citation.title
Revision 1.5Jul 18, 2018Group: Data collection / Category: em_software / Item: _em_software.name
Revision 1.6Dec 18, 2019Group: Author supporting evidence / Other / Category: atom_sites / pdbx_audit_support
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] / _pdbx_audit_support.funding_organization

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Structure visualization

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Assembly

Deposited unit
A: DNA (5'-D(*GP*GP*GP*CP*TP*AP*AP*TP*GP*AP*CP*GP*GP*CP*GP*AP*AP*TP*AP*CP*CP*C)-3')
B: DNA (29-MER)
R: RNA (5'-R(P*CP*GP*GP*AP*GP*AP*GP*GP*UP*A)-3')
G: DNA-directed RNA polymerase subunit alpha
H: DNA-directed RNA polymerase subunit alpha
I: DNA-directed RNA polymerase subunit beta
J: DNA-directed RNA polymerase subunit beta'
K: DNA-directed RNA polymerase subunit omega
hetero molecules


Theoretical massNumber of molelcules
Total (without water)392,52011
Polymers392,3648
Non-polymers1553
Water0
1


TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area40040 Å2
ΔGint-207 kcal/mol
Surface area142140 Å2

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Components

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DNA chain , 2 types, 2 molecules AB

#1: DNA chain DNA (5'-D(*GP*GP*GP*CP*TP*AP*AP*TP*GP*AP*CP*GP*GP*CP*GP*AP*AP*TP*AP*CP*CP*C)-3')


Mass: 8840.689 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Enterobacteria phage T7 (bacteriophage)
#2: DNA chain DNA (29-MER)


Mass: 8813.646 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Enterobacteria phage T7 (bacteriophage)

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RNA chain , 1 types, 1 molecules R

#3: RNA chain RNA (5'-R(P*CP*GP*GP*AP*GP*AP*GP*GP*UP*A)-3')


Mass: 6509.968 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Enterobacteria phage T7 (bacteriophage)

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DNA-directed RNA polymerase subunit ... , 4 types, 5 molecules GHIJK

#4: Protein DNA-directed RNA polymerase subunit alpha / Polymerase / RNAP subunit alpha / RNA polymerase subunit alpha / Transcriptase subunit alpha


Mass: 26459.125 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (strain K12) (bacteria)
Strain: K12 / Gene: rpoA, pez, phs, sez, b3295, JW3257 / Production host: Escherichia coli (E. coli) / References: UniProt: P0A7Z4, DNA-directed RNA polymerase
#5: 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)
Strain: K12
Gene: rpoB, groN, nitB, rif, ron, stl, stv, tabD, b3987, JW3950
Production host: Escherichia coli (E. coli) / References: UniProt: P0A8V2, DNA-directed RNA polymerase
#6: 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)
Strain: K12 / Gene: rpoC, tabB, b3988, JW3951 / Production host: Escherichia coli (E. coli) / References: UniProt: P0A8T7, DNA-directed RNA polymerase
#7: Protein DNA-directed RNA polymerase subunit omega / Polymerase / RNAP omega subunit / RNA polymerase omega subunit / Transcriptase subunit omega


Mass: 9094.239 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (strain K12) (bacteria)
Strain: K12 / Gene: rpoZ, b3649, JW3624 / Production host: Escherichia coli (E. coli) / References: UniProt: P0A800, DNA-directed RNA polymerase

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Non-polymers , 2 types, 3 molecules

#8: Chemical ChemComp-MG / MAGNESIUM ION / Magnesium


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg
#9: Chemical ChemComp-ZN / ZINC ION / Zinc


Mass: 65.409 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Zn

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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: E.coli RNA polymerase elongation complex / Type: COMPLEX / Entity ID: #1-#8 / Source: RECOMBINANT
Molecular weightValue: 0.393 MDa / Experimental value: NO
Source (natural)Organism: Escherichia coli (E. coli)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 8 / Details: 20 mM Tris pH8.0, 150 mM KCl, 5mM MgCl2, 5mM DTT
SpecimenConc.: 3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 400 divisions/in. / Grid type: C-flat CF1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 295.15 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
Image recordingElectron dose: 71 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.12_2829: / Classification: refinement
EM software
IDNameVersionCategory
7UCSF Chimeramodel fitting
12RELION2.0beta3D reconstruction
13PHENIXmodel refinement
CTF correctionDetails: Used CTFFIND4 / Type: NONE
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 4.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 162500 / Num. of class averages: 2 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Refine LS restraints
Refinement-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00726526
ELECTRON MICROSCOPYf_angle_d1.02736085
ELECTRON MICROSCOPYf_dihedral_angle_d8.38516111
ELECTRON MICROSCOPYf_chiral_restr0.0664128
ELECTRON MICROSCOPYf_plane_restr0.0074511

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