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- PDB-8ehf: Cryo-EM structure of his-elemental paused elongation complex with... -

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Basic information

Entry
Database: PDB / ID: 8ehf
TitleCryo-EM structure of his-elemental paused elongation complex with an unfolded TL (1)
Components
  • (DNA-directed RNA polymerase subunit ...) x 4
  • RNA
  • non-template DNA
  • template DNA
KeywordsTRANSCRIPTION / TRANSFERASE/DNA/RNA / RNA polymerase / cryo-EM structure / elemental pause / Escherichia coli / TRANSFERASE-DNA-RNA complex / transcriptional regulation / transcriptional pausing
Function / homology
Function and homology information


DNA-directed RNA polymerase complex / ribonucleoside binding / DNA-directed 5'-3' RNA polymerase activity / DNA-directed RNA polymerase / protein dimerization activity / DNA-templated transcription / magnesium ion binding / DNA binding / zinc ion binding / cytoplasm
Similarity search - Function
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 ...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 / RPB6/omega subunit-like superfamily / RNA polymerase Rpb1, clamp domain superfamily / RNA polymerase Rpb2, domain 2 superfamily / RNA polymerase Rpb1, domain 3 / RNA polymerase Rpb1, domain 3 / DNA-directed RNA polymerase, subunit beta-prime / RNA polymerase Rpb1, domain 1 / RNA polymerase Rpb1, domain 1 / 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 / RNA polymerase, alpha subunit / RNA polymerase Rpb1, domain 5 / RNA polymerase Rpb1, domain 4 / RNA polymerase Rpb1, domain 2 / RNA polymerase Rpb1, domain 5 / RNA polymerase Rpb1, domain 4 / RNA polymerase, beta subunit, protrusion / RNA polymerase beta subunit / RNA polymerase, N-terminal / RNA polymerase Rpb1, funnel domain superfamily / RNA polymerase I subunit A N-terminus / 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
Similarity search - Domain/homology
Chem-4QM / DNA / DNA (> 10) / RNA / RNA (> 10) / DNA-directed RNA polymerase subunit beta' / DNA-directed RNA polymerase subunit alpha / DNA-directed RNA polymerase subunit omega / DNA-directed RNA polymerase subunit beta
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsKang, J.Y. / Chen, J. / Llewellyn, E. / Landick, R. / Darst, S.A.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM118130 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM38330 United States
CitationJournal: Proc Natl Acad Sci U S A / Year: 2023
Title: An ensemble of interconverting conformations of the elemental paused transcription complex creates regulatory options.
Authors: Jin Young Kang / Tatiana V Mishanina / Yu Bao / James Chen / Eliza Llewellyn / James Liu / Seth A Darst / Robert Landick /
Abstract: Transcriptional pausing underpins the regulation of cellular RNA synthesis, but its mechanism remains incompletely understood. Sequence-specific interactions of DNA and RNA with the dynamic, ...Transcriptional pausing underpins the regulation of cellular RNA synthesis, but its mechanism remains incompletely understood. Sequence-specific interactions of DNA and RNA with the dynamic, multidomain RNA polymerase (RNAP) trigger reversible conformational changes at pause sites that temporarily interrupt the nucleotide addition cycle. These interactions initially rearrange the elongation complex (EC) into an elemental paused EC (ePEC). ePECs can form longer-lived PECs by further rearrangements or interactions of diffusible regulators. For both bacterial and mammalian RNAPs, a half-translocated state in which the next DNA template base fails to load into the active site appears central to the ePEC. Some RNAPs also swivel interconnected modules that may stabilize the ePEC. However, it is unclear whether swiveling and half-translocation are requisite features of a single ePEC state or if multiple ePEC states exist. Here, we use cryo-electron microscopy (cryo-EM) analysis of ePECs with different RNA-DNA sequences combined with biochemical probes of ePEC structure to define an interconverting ensemble of ePEC states. ePECs occupy either pre- or half-translocated states but do not always swivel, indicating that difficulty in forming the posttranslocated state at certain RNA-DNA sequences may be the essence of the ePEC. The existence of multiple ePEC conformations has broad implications for transcriptional regulation.
History
DepositionSep 14, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 1, 2023Provider: repository / Type: Initial release
Revision 1.1Jun 19, 2024Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond / em_3d_fitting_list
Item: _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id ..._em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: non-template DNA
B: template DNA
R: RNA
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)395,49212
Polymers394,9598
Non-polymers5344
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration, a gel filtration chromatography combined with SDS-PAGE confirmed the presence of all the RNA polymerase subunits
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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DNA chain , 2 types, 2 molecules AB

#1: DNA chain non-template DNA


Mass: 9881.364 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Escherichia coli (E. coli)
#2: DNA chain template DNA


Mass: 9706.256 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Escherichia coli (E. coli)

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RNA chain , 1 types, 1 molecules R

#3: RNA chain RNA


Mass: 6047.593 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Escherichia coli (E. coli)

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

#4: Protein DNA-directed RNA polymerase subunit alpha / 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 (E. coli) / Gene: rpoA, Z4665, ECs4160 / Production host: Escherichia coli (E. coli) / References: UniProt: P0A7Z6, DNA-directed RNA polymerase
#5: Protein DNA-directed RNA polymerase subunit beta / 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 (E. coli) / Gene: rpoB, Z5560, ECs4910 / Production host: Escherichia coli (E. coli) / References: UniProt: P0A8V4, DNA-directed RNA polymerase
#6: Protein DNA-directed RNA polymerase subunit beta' / RNAP subunit beta' / RNA polymerase subunit beta' / Transcriptase subunit beta'


Mass: 155334.719 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Production host: Escherichia coli (E. coli) / References: UniProt: C3SIA2, DNA-directed RNA polymerase
#7: Protein DNA-directed RNA polymerase subunit omega / 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 (E. coli) / Gene: rpoZ, Z5075, ECs4524 / Production host: Escherichia coli (E. coli) / References: UniProt: P0A802, DNA-directed RNA polymerase

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Non-polymers , 3 types, 4 molecules

#8: Chemical ChemComp-4QM / (3R,5S,7R,8R,9S,10S,12S,13R,14S,17R)-10,13-dimethyl-17-[(2R)-pentan-2-yl]-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3,7,12-triol


Mass: 378.588 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C24H42O3
#9: Chemical ChemComp-MG / MAGNESIUM ION


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


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

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Details

Has ligand of interestN

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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: his-elemental paused elongation complex / Type: COMPLEX
Details: An RNA polymerase elongation complex was assembled at the weak elemental pausing site derived from his-pause sequence.
Entity ID: #1-#7 / Source: MULTIPLE SOURCES
Molecular weightValue: 0.45 MDa / Experimental value: NO
Source (natural)Organism: Escherichia coli (E. coli)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMTris1
2150 mMpotassium glutamate1
35 mMmagnesium chlorideMgCl21
45 mMdithiothreitol1
SpecimenConc.: 7 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-1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 295 K / Details: blot force 0, blot time 3.5 s

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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 FIELD / Nominal defocus max: 2400 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm
Specimen holderSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 10 sec. / Electron dose: 47.34 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 6939
Image scansWidth: 3710 / Height: 3838 / Movie frames/image: 50 / Used frames/image: 1-50

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Processing

SoftwareName: PHENIX / Version: 1.19.2_4158: / Classification: refinement
EM software
IDNameVersionCategory
1Gautomatchparticle selection
2SerialEMimage acquisition
4GctfCTF correction
7UCSF Chimeramodel fitting
9PHENIXmodel refinement
10cryoSPARC2initial Euler assignment
11RELIONfinal Euler assignment
12RELIONclassification
13RELION3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 2126200
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 432300 / Algorithm: BACK PROJECTION / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Atomic model building

3D fitting-ID: 1 / Accession code: 6C6T / Initial refinement model-ID: 1 / PDB-ID: 6C6T

6c6t

/ Source name: PDB / Type: experimental model

IDPdb chain-IDPdb chain residue range
1G7-234
2H4-232
3I2-1342
4J16-1373
5K2-84
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00526626
ELECTRON MICROSCOPYf_angle_d0.62836235
ELECTRON MICROSCOPYf_dihedral_angle_d17.89410306
ELECTRON MICROSCOPYf_chiral_restr0.0454153
ELECTRON MICROSCOPYf_plane_restr0.0044520

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