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- PDB-6asx: CryoEM structure of E.coli his pause elongation complex -

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Basic information

Entry
Database: PDB / ID: 6asx
TitleCryoEM structure of E.coli his pause elongation complex
Components
  • (DNA (32-MER)) x 2
  • (DNA-directed RNA polymerase subunit ...Polymerase) x 4
  • RNA (29-MER)
Keywordstranscription/dna/rna / DNA-dependent RNA polymerase / TRANSCRIPTION / transcription-dna-rna complex
Function / homologyRNA polymerase subunit, RPB6/omega / RPB6/omega subunit-like superfamily / 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 beta subunit / DNA-directed RNA polymerase, subunit beta-prime / RNA polymerase Rpb2, OB-fold / DNA-directed RNA polymerase, subunit 2 ...RNA polymerase subunit, RPB6/omega / RPB6/omega subunit-like superfamily / 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 beta subunit / DNA-directed RNA polymerase, subunit beta-prime / RNA polymerase Rpb2, OB-fold / DNA-directed RNA polymerase, subunit 2 / DNA-directed RNA polymerase, beta subunit, external 1 domain / RNA polymerase, RBP11-like subunit / RNA polymerase Rpb2, domain 3 / 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 Rpb2, domain 6 / RNA polymerase Rpb1, domain 2 / RNA polymerase Rpb3/RpoA insert domain / RNA polymerase Rpb6 / RNA polymerase Rpb3/Rpb11 dimerisation domain / Bacterial RNA polymerase, alpha chain C terminal domain / DNA-directed RNA polymerase beta subunit, bacterial-type / RNA polymerase, beta subunit, protrusion / RNA polymerase Rpb2, domain 2 / DNA-directed RNA polymerase, omega subunit / RNA polymerases beta chain signature. / RNA polymerase beta subunit external 1 domain / RNA polymerase Rpb1, domain 4 / RNA polymerase Rpb1, domain 5 / RNA polymerase Rpb1, domain 1 / RNA polymerase Rpb1, domain 3 / RNA polymerase Rpb2, domain 3 / RNA polymerase Rpb2, domain 2 / RNA polymerase, alpha subunit / RNA polymerase, subunit omega/K/RPB6 / RNA polymerase, N-terminal / RNA polymerase Rpb1, domain 3 / RNA polymerase Rpb1, domain 1 / 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 Rpb2, domain 7 / RNA polymerase Rpb2, domain 7 / 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 / DNA binding / zinc ion binding / membrane / cytosol / cytoplasm / DNA-directed RNA polymerase subunit alpha / DNA-directed RNA polymerase subunit omega / DNA-directed RNA polymerase subunit beta' / DNA-directed RNA polymerase subunit beta
Function and homology information
Specimen sourceEscherichia coli (E. coli)
Salmonella enterica subsp. enterica serovar Typhimurium (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / 3.8 Å resolution
AuthorsKang, J.Y. / Landick, R. / Darst, S.A.
CitationJournal: Mol. Cell / Year: 2018
Title: RNA Polymerase Accommodates a Pause RNA Hairpin by Global Conformational Rearrangements that Prolong Pausing.
Authors: Jin Young Kang / Tatiana V Mishanina / Michael J Bellecourt / Rachel Anne Mooney / Seth A Darst / Robert Landick
Abstract: Sequence-specific pausing by RNA polymerase (RNAP) during transcription plays crucial and diverse roles in gene expression. In bacteria, RNA structures are thought to fold within the RNA exit channel ...Sequence-specific pausing by RNA polymerase (RNAP) during transcription plays crucial and diverse roles in gene expression. In bacteria, RNA structures are thought to fold within the RNA exit channel of the RNAP and can increase pause lifetimes significantly. The biophysical mechanism of pausing is uncertain. We used single-particle cryo-EM to determine structures of paused complexes, including a 3.8-Å structure of an RNA hairpin-stabilized, paused RNAP that coordinates RNA folding in the his operon attenuation control region of E. coli. The structures revealed a half-translocated pause state (RNA post-translocated, DNA pre-translocated) that can explain transcriptional pausing and a global conformational change of RNAP that allosterically inhibits trigger loop folding and can explain pause hairpin action. Pause hairpin interactions with the RNAP RNA exit channel suggest how RNAP guides the formation of nascent RNA structures.
Validation Report
SummaryFull reportAbout validation report
DateDeposition: Aug 25, 2017 / Release: Mar 28, 2018
RevisionDateData content typeGroupCategoryItemProviderType
1.0Mar 28, 2018Structure modelrepositoryInitial release
1.1Jul 18, 2018Structure modelData collectionem_software_em_software.name

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Structure visualization

Movie
  • Deposited structure unit
  • Imaged by Jmol
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  • Superimposition on EM map
  • EMDB-7002
  • Imaged by UCSF Chimera
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Assembly

Deposited unit
A: DNA (32-MER)
B: DNA (32-MER)
R: RNA (29-MER)
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)397,57411
Polyers397,4198
Non-polymers1553
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551
Buried area (Å2)39740
ΔGint (kcal/M)-262
Surface area (Å2)149560

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Components

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DNA chain , 2 types, 2 molecules AB

#1: DNA chain DNA (32-MER) / Non-template DNA


Mass: 9881.364 Da / Num. of mol.: 1 / Details: no template DNA
Source: (synth.) Salmonella enterica subsp. enterica serovar Typhimurium (bacteria)
#2: DNA chain DNA (32-MER) / template DNA


Mass: 9706.256 Da / Num. of mol.: 1 / Details: template DNA
Source: (synth.) Salmonella enterica subsp. enterica serovar Typhimurium (bacteria)

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RNA chain , 1 types, 1 molecules R

#3: RNA chain RNA (29-MER)


Mass: 9231.442 Da / Num. of mol.: 1
Source: (synth.) Salmonella enterica subsp. enterica serovar Typhimurium (bacteria)

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

#4: Protein/peptide 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: (gene. exp.) Escherichia coli (E. coli) / Strain: K12 / Gene: rpoA, pez, phs, sez, b3295, JW3257 / Production host: Escherichia coli (E. coli) / References: UniProt: P0A7Z4, DNA-directed RNA polymerase
#5: Protein/peptide 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: (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/peptide 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: (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/peptide DNA-directed RNA polymerase subunit omega / Polymerase / RNAP omega subunit / RNA polymerase omega subunit / Transcriptase subunit omega


Mass: 9493.681 Da / Num. of mol.: 1 / Source: (gene. exp.) Escherichia coli (E. coli) / 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


Mass: 24.305 Da / Num. of mol.: 1 / Formula: Mg / Magnesium
#9: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 2 / Formula: Zn / Zinc

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / Reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: E.coli his pause elongation complex / Type: COMPLEX / Entity ID: 1, 2, 3, 4, 5, 6, 7 / Source: MULTIPLE SOURCES
Molecular weightValue: 0.4 MDa / Experimental value: NO
Source (natural)Organism: Escherichia coli (E. coli)
Buffer solutionDetails: 20 mM Tris pH8.0, 150 mM potassium glutamate, 5 mM MgCl2, 5 mM DTT
pH: 8
SpecimenConc.: 5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 400 / Grid type: C-flat
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 295 kelvins

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyMicroscope model: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 29500 / Nominal defocus max: 2400 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm / C2 aperture diameter: 70 microns
Specimen holderSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 10 sec. / Electron dose: 72.4 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Number of grids imaged: 2 / Number of real images: 2910
Image scansWidth: 3710 / Height: 3838 / Movie frames/image: 50 / Used frames/image: 3-50

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Processing

SoftwareName: PHENIX / Version: 1.12_2829: / Classification: refinement
EM software
IDNameVersionCategory
2SerialEMimage acquisition
4CTFFIND4.0CTF correction
7UCSF Chimera1.11model fitting
9RELION2.0initial Euler assignment
10RELION2.0final Euler assignment
11RELION2.0classification
12RELION2.03D reconstruction
13PHENIX1.11model refinement
CTF correctionType: NONE
Particle selectionNumber of particles selected: 408000
SymmetryPoint symmetry: C1
3D reconstructionResolution: 3.8 Å / Resolution method: FSC 0.143 CUT-OFF / Number of particles: 171300 / Number of class averages: 1 / Symmetry type: POINT
Atomic model buildingRef protocol: RIGID BODY FIT / Ref space: REAL
Refine LS restraints
Refine IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00826896
ELECTRON MICROSCOPYf_angle_d1.03236638
ELECTRON MICROSCOPYf_dihedral_angle_d10.64516312
ELECTRON MICROSCOPYf_chiral_restr0.0684203
ELECTRON MICROSCOPYf_plane_restr0.0084541

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