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- PDB-7m8e: E.coli RNAP-RapA elongation complex -

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Basic information

Entry
Database: PDB / ID: 7m8e
TitleE.coli RNAP-RapA elongation complex
Components
  • (DNA-directed RNA polymerase subunit ...) x 4
  • Nontemplate DNA (39-MER)
  • RNA (5'-R(P*AP*UP*CP*GP*GP*CP*UP*CP*A)-3')
  • RNA polymerase-associated protein RapA
  • Template DNA (39-MER)
KeywordsTRANSFERASE/HYDROLASE/DNA/RNA / Swi2/snf2 / RapA / ATPase / RNA polymerase / TRANSFERASE-HYDROLASE-DNA-RNA complex
Function / homology
Function and homology information


hydrolase activity, acting on acid anhydrides / DNA-directed RNA polymerase complex / helicase activity / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / ribonucleoside binding / DNA-directed RNA polymerase / DNA-directed RNA polymerase activity / protein dimerization activity / DNA-templated transcription / regulation of DNA-templated transcription ...hydrolase activity, acting on acid anhydrides / DNA-directed RNA polymerase complex / helicase activity / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / ribonucleoside binding / DNA-directed RNA polymerase / DNA-directed RNA polymerase activity / protein dimerization activity / DNA-templated transcription / regulation of DNA-templated transcription / magnesium ion binding / DNA binding / zinc ion binding / ATP binding
Similarity search - Function
: / RNA polymerase recycling, bacterial, C-terminal / RNA polymerase-associated protein RapA / RapA, N-terminal Tudor like domain 1 / RapA, N-terminal Tudor-like domain 2 / RNA polymerase recycling family C-terminal / RapA N-terminal Tudor like domain / RapA N-terminal Tudor like domain 1 / RNA polymerase Rpb1 funnel domain / RNA polymerase II/Efflux pump adaptor protein, barrel-sandwich hybrid domain ...: / RNA polymerase recycling, bacterial, C-terminal / RNA polymerase-associated protein RapA / RapA, N-terminal Tudor like domain 1 / RapA, N-terminal Tudor-like domain 2 / RNA polymerase recycling family C-terminal / RapA N-terminal Tudor like domain / RapA N-terminal Tudor like domain 1 / RNA polymerase Rpb1 funnel domain / RNA polymerase II/Efflux pump adaptor protein, barrel-sandwich hybrid domain / Topoisomerase I; Chain A, domain 4 / DNA-directed RNA polymerase, insert domain / RNA Polymerase Alpha Subunit; Chain A, domain 2 / : / SNF2-like, N-terminal domain superfamily / SNF2, N-terminal / SNF2-related domain / 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 beta subunit, bacterial-type / DNA-directed RNA polymerase, alpha subunit / Beta Complex / RNA polymerase Rpb6 / Helicase conserved C-terminal domain / RNA polymerase, subunit omega/Rpo6/RPB6 / RNA polymerase Rpb6 / RNA polymerase Rpb2, domain 2 superfamily / RNA polymerase Rpb1, domain 3 superfamily / RPB6/omega subunit-like superfamily / 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 Rpb1, domain 3 / RNA polymerase Rpb1, domain 3 / RNA polymerase Rpb1, domain 1 / RNA polymerase Rpb1, domain 1 / RNA polymerase, beta subunit, protrusion / RNA polymerase beta subunit / 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 / DNA-directed RNA polymerase, insert domain superfamily / RNA polymerase, N-terminal / RNA polymerase Rpb2, domain 2 / RNA polymerase Rpb1, funnel domain superfamily / RNA polymerase Rpb2, domain 2 / RNA polymerase I subunit A N-terminus / RNA polymerase, RBP11-like subunit / 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 / OB fold (Dihydrolipoamide Acetyltransferase, E2P) / helicase superfamily c-terminal domain / Superfamilies 1 and 2 helicase C-terminal domain profile. / Superfamilies 1 and 2 helicase ATP-binding type-1 domain profile. / DEAD-like helicases superfamily / Helicase, C-terminal / Helicase superfamily 1/2, ATP-binding domain / Beta Barrel / P-loop containing nucleoside triphosphate hydrolase / Orthogonal Bundle / Mainly Beta / Mainly Alpha
Similarity search - Domain/homology
DNA / DNA (> 10) / RNA / DNA-directed RNA polymerase subunit alpha / RNA polymerase-associated protein RapA / DNA-directed RNA polymerase subunit beta' / 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: 3.4 Å
AuthorsShi, W. / Liu, B.
CitationJournal: Nucleic Acids Res / Year: 2021
Title: Structural basis for activation of Swi2/Snf2 ATPase RapA by RNA polymerase.
Authors: Wei Shi / Wei Zhou / Ming Chen / Yang Yang / Yangbo Hu / Bin Liu /
Abstract: RapA is a bacterial RNA polymerase (RNAP)-associated Swi2/Snf2 ATPase that stimulates RNAP recycling. The ATPase activity of RapA is autoinhibited by its N-terminal domain (NTD) but activated with ...RapA is a bacterial RNA polymerase (RNAP)-associated Swi2/Snf2 ATPase that stimulates RNAP recycling. The ATPase activity of RapA is autoinhibited by its N-terminal domain (NTD) but activated with RNAP bound. Here, we report a 3.4-Å cryo-EM structure of Escherichia coli RapA-RNAP elongation complex, in which the ATPase active site of RapA is structurally remodeled. In this process, the NTD of RapA is wedged open by RNAP β' zinc-binding domain (ZBD). In addition, RNAP β flap tip helix (FTH) forms extensive hydrophobic interactions with RapA ATPase core domains. Functional assay demonstrates that removing the ZBD or FTH of RNAP significantly impairs its ability to activate the ATPase activity of RapA. Our results provide the structural basis of RapA ATPase activation by RNAP, through the active site remodeling driven by the ZBD-buttressed large-scale opening of NTD and the direct interactions between FTH and ATPase core domains.
History
DepositionMar 29, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Aug 18, 2021Provider: repository / Type: Initial release
Revision 1.1Sep 8, 2021Group: Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed ..._citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID
Revision 1.2Oct 20, 2021Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.3May 29, 2024Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_3d_fitting_list / pdbx_initial_refinement_model
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

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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
F: RNA polymerase-associated protein RapA
1: Nontemplate DNA (39-MER)
2: Template DNA (39-MER)
3: RNA (5'-R(P*AP*UP*CP*GP*GP*CP*UP*CP*A)-3')
hetero molecules


Theoretical massNumber of molelcules
Total (without water)528,42912
Polymers528,2749
Non-polymers1553
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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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 / 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 (E. coli) / Gene: rpoA / Production host: Escherichia coli (E. coli)
References: UniProt: A0A073G207, DNA-directed RNA polymerase
#2: 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
#3: Protein DNA-directed RNA polymerase subunit beta' / RNAP subunit beta' / RNA polymerase subunit beta' / Transcriptase subunit beta'


Mass: 156537.031 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: rpoC / Production host: Escherichia coli (E. coli) / References: UniProt: D8ED86, DNA-directed RNA polymerase
#4: 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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DNA chain , 2 types, 2 molecules 12

#6: DNA chain Nontemplate DNA (39-MER)


Mass: 11949.682 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Escherichia coli (E. coli)
#7: DNA chain Template DNA (39-MER)


Mass: 12040.719 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Escherichia coli (E. coli)

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Protein / RNA chain , 2 types, 2 molecules F3

#5: Protein RNA polymerase-associated protein RapA / ATP-dependent helicase HepA


Mass: 110726.789 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: rapA / Production host: Escherichia coli (E. coli)
References: UniProt: C3TR27, Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement
#8: RNA chain RNA (5'-R(P*AP*UP*CP*GP*GP*CP*UP*CP*A)-3')


Mass: 2831.743 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Escherichia coli (E. coli)

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Non-polymers , 2 types, 3 molecules

#9: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Zn / Feature type: SUBJECT OF INVESTIGATION
#10: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION

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Details

Has ligand of interestY

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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: RNAP-RapA elongation complex / Type: COMPLEX / Entity ID: #1-#8 / Source: MULTIPLE SOURCES
Molecular weightValue: 0.53 MDa / Experimental value: YES
Source (natural)Organism: Escherichia coli (E. coli)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
SpecimenConc.: 5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R2/2
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 296 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 FIELD / Nominal magnification: 96000 X / Nominal defocus max: 2400 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm / Alignment procedure: BASIC
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 32 sec. / Electron dose: 40 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON III (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 3218
Image scansWidth: 4096 / Height: 4096

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Processing

EM software
IDNameVersionCategory
1EPUimage acquisition
12UCSF Chimeramodel fitting
19cryoSPARC2.15particle selection
20cryoSPARC2.15CTF correction
21cryoSPARC2.15initial Euler assignment
22cryoSPARC2.15final Euler assignment
23cryoSPARC2.15classification
24cryoSPARC2.153D reconstruction
25PHENIXmodel refinement
CTF correctionType: NONE
Particle selectionNum. of particles selected: 1456920
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 58761 / Num. of class averages: 24 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Atomic model buildingPDB-ID: 4S20

4s20


Accession code: 4S20 / Source name: PDB / Type: experimental model

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