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- PDB-8t0l: E. coli Sw2/Snf2 ATPase RapA bound to both ADP-AlF3 and reconstit... -

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Basic information

Entry
Database: PDB / ID: 8t0l
TitleE. coli Sw2/Snf2 ATPase RapA bound to both ADP-AlF3 and reconstituted E. coli RNA polymerase post-termination complex on negatively-supercoiled DNA
Components
  • (DNA-directed RNA polymerase subunit ...) x 4
  • DNA (29-MER)
  • DNA (5'-D(P*TP*CP*TP*GP*AP*AP*TP*TP*TP*AP*AP*AP*TP*TP*CP*AP*GP*A)-3')
  • RNA polymerase-associated protein RapA
KeywordsTRANSCRIPTION/DNA / RNA polymerase / Negatively supercoiled DNA / Sigma-independent transcription / RapA / RNAP recycling / Sw2/Snf2 ATPase / TRANSCRIPTION-DNA 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 / cytosol / cytoplasm
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 / : / SNF2-like, N-terminal domain superfamily / SNF2, N-terminal ...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 / : / 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 / DNA-directed RNA polymerase, subunit beta-prime / 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, clamp domain superfamily / RNA polymerase Rpb1, domain 3 / RNA polymerase Rpb1, domain 3 / RNA polymerase Rpb1, domain 1 / RNA polymerase, beta subunit, protrusion / RNA polymerase beta subunit / RNA polymerase Rpb1, domain 1 / 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 Rpb2, domain 2 / RNA polymerase Rpb2, domain 2 / RNA polymerase, N-terminal / RNA polymerase Rpb1, funnel domain superfamily / 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 / 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 / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
ADENOSINE-5'-DIPHOSPHATE / ALUMINUM FLUORIDE / DNA / DNA (> 10) / DNA-directed RNA polymerase subunit beta' / DNA-directed RNA polymerase subunit omega / RNA polymerase-associated protein RapA / DNA-directed RNA polymerase subunit beta / DNA-directed RNA polymerase subunit alpha
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.62 Å
AuthorsBrewer, J.J. / Darst, S.A. / Campbell, E.A.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS) United States
CitationJournal: Nat Struct Mol Biol / Year: 2025
Title: RapA opens the RNA polymerase clamp to disrupt post-termination complexes and prevent cytotoxic R-loop formation.
Authors: Joshua J Brewer / Koe Inlow / Rachel A Mooney / Barbara Bosch / Paul Dominic B Olinares / Leandro Pimentel Marcelino / Brian T Chait / Robert Landick / Jeff Gelles / Elizabeth A Campbell / Seth A Darst /
Abstract: Following transcript release during intrinsic termination, Escherichia coli RNA polymerase (RNAP) often remains associated with DNA in a post-termination complex (PTC). RNAPs in PTCs are removed from ...Following transcript release during intrinsic termination, Escherichia coli RNA polymerase (RNAP) often remains associated with DNA in a post-termination complex (PTC). RNAPs in PTCs are removed from the DNA by the SWI2/SNF2 adenosine triphosphatase (ATPase) RapA. Here we determined PTC structures on negatively supercoiled DNA and with RapA engaged to dislodge the PTC. We found that core RNAP in the PTC can unwind DNA and initiate RNA synthesis but is prone to producing R-loops. Nucleotide binding to RapA triggers a conformational change that opens the RNAP clamp, allowing DNA in the RNAP cleft to reanneal and dissociate. We show that RapA helps to control cytotoxic R-loop formation in vivo, likely by disrupting PTCs. We suggest that analogous ATPases acting on PTCs to suppress transcriptional noise and R-loop formation may be widespread. These results hold importance for the bacterial transcription cycle and highlight a role for RapA in maintaining genome stability.
History
DepositionJun 1, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Apr 2, 2025Provider: repository / Type: Initial release
Revision 1.1Apr 30, 2025Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
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
F: RNA polymerase-associated protein RapA
A: DNA (29-MER)
B: DNA (5'-D(P*TP*CP*TP*GP*AP*AP*TP*TP*TP*AP*AP*AP*TP*TP*CP*AP*GP*A)-3')
hetero molecules


Theoretical massNumber of molelcules
Total (without water)484,28513
Polymers483,6198
Non-polymers6665
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

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

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


Mass: 25597.117 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli)
Gene: rpoA, A5U30_004529, A8502_004046, ACN81_25405, ACU57_21075, AW118_24785, AWP47_12760, B6R15_004092, B6R31_004991, BANRA_01926, BE932_15420, BER14_25075, BG944_001110, BGM66_003616, BGZ_02613, ...Gene: rpoA, A5U30_004529, A8502_004046, ACN81_25405, ACU57_21075, AW118_24785, AWP47_12760, B6R15_004092, B6R31_004991, BANRA_01926, BE932_15420, BER14_25075, BG944_001110, BGM66_003616, BGZ_02613, BGZ_04102, BJI68_08790, BK292_20390, BK383_24760, BTB68_004379, BTQ06_10575, BvCmsKKP061_03007, BvCmsSIP010_02662, BXT93_05590, C0P57_002645, C1Q91_004927, C2121_004185, C2R31_004610, C3F40_19835, C9Z68_22520, CF22_004872, CG704_20985, CG831_003626, CIG67_10790, CTR35_003607, CV83915_02803, D3G36_23165, D4M65_20540, D4N09_20140, D9D43_22665, D9E49_05125, D9J61_16620, DD762_23515, DIV22_15290, DNQ45_03725, DNX30_25510, DS732_24230, DTL43_21030, DU321_11970, E4K51_21460, E5H86_24555, E6D34_21790, EAI46_07445, ECs4160, EIA08_23925, EIZ93_12500, EN85_004372, EPS76_07245, EPS97_20125, EWK56_24965, ExPECSC038_03663, F7F11_22755, F7N46_24040, F9413_21265, F9461_25685, F9B07_24715, FGAF848_26080, FIJ20_21225, FJQ40_18275, FKO60_25605, FOI11_019290, FOI11_03890, FORC44_0502, FPI65_20275, FPS11_25460, FVB16_04840, FZU14_21790, G3V95_19815, G3W53_20925, G4A38_21480, G4A47_20750, G5603_24555, GAI89_24860, GAJ12_24750, GKF66_21515, GNW61_16600, GOP25_22675, GP711_23275, GP954_00975, GP975_01155, GP979_02035, GQA06_03980, GQE86_20050, GQM04_10535, GQM13_25170, GQM21_11200, GQN34_23235, GQW07_21580, GRC73_21780, GRO95_20845, GRW05_09030, GRW24_04785, GRW56_00265, GRW57_03570, GUC01_21150, H0O72_19385, HEP30_018605, HEP34_004777, HHH44_004542, HI055_004133, HIE29_005180, HJQ60_005018, HLX92_10105, HLZ50_22210, HMV95_19575, HV109_02220, HV209_14745, HVV39_09235, HVW04_17565, HVW43_18700, HVY77_02205, I6H00_20955, I6H02_11960, IFB95_004799, J0541_004390, JNP96_25275, KV499_004428, NCTC10082_02846, NCTC10089_00509, NCTC10418_00698, NCTC10767_01523, NCTC10865_00668, NCTC10974_00559, NCTC11126_02779, NCTC11181_02745, NCTC11341_01882, NCTC13148_03434, NCTC7927_00558, NCTC7928_02508, NCTC8009_01520, NCTC8179_05950, NCTC8333_00496, NCTC8500_00344, NCTC8621_00515, NCTC8622_00499, NCTC8959_03254, NCTC8960_03077, NCTC9044_01349, NCTC9077_00611, NCTC9081_00926, NCTC9117_00762, NCTC9702_00544, NCTC9706_02744, NEP60_26260, OGM49_22300, QDW62_02225, RZR61_12860, SAMEA3472112_00700, SAMEA3752557_01945, WR15_19575
Production host: Escherichia coli (E. coli) / References: UniProt: C3SR67, DNA-directed RNA polymerase
#2: Protein DNA-directed RNA polymerase subunit beta


Mass: 150560.562 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: rpoB / Production host: Escherichia coli (E. coli) / References: UniProt: C3SIA7
#3: Protein DNA-directed RNA polymerase subunit beta' / RNAP subunit beta' / RNA polymerase subunit beta' / Transcriptase subunit beta'


Mass: 150406.328 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: rpoC, C2R31_004866 / Production host: Escherichia coli (E. coli)
References: UniProt: A0A369F490, DNA-directed RNA polymerase
#4: Protein DNA-directed RNA polymerase subunit omega / RNAP omega subunit / RNA polymerase omega subunit / Transcriptase subunit omega


Mass: 8250.298 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: rpoZ, EcE24377A_4152 / Production host: Escherichia coli (E. coli) / References: UniProt: A7ZTK1, DNA-directed RNA polymerase

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Protein , 1 types, 1 molecules F

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


Mass: 109096.914 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: rapA, ECS88_0062 / Production host: Escherichia coli (E. coli)
References: UniProt: B7MAI2, Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement

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DNA chain , 2 types, 2 molecules AB

#6: DNA chain DNA (29-MER)


Mass: 7158.800 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#7: DNA chain DNA (5'-D(P*TP*CP*TP*GP*AP*AP*TP*TP*TP*AP*AP*AP*TP*TP*CP*AP*GP*A)-3')


Mass: 6951.477 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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Non-polymers , 4 types, 5 molecules

#8: Chemical ChemComp-MG / MAGNESIUM ION


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


Mass: 65.409 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Zn
#10: Chemical ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE


Mass: 427.201 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Feature type: SUBJECT OF INVESTIGATION / Comment: ADP, energy-carrying molecule*YM
#11: Chemical ChemComp-AF3 / ALUMINUM FLUORIDE


Mass: 83.977 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: AlF3 / Feature type: SUBJECT OF INVESTIGATION

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Details

Has ligand of interestY
Has protein modificationN

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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 Post-termination complex with open DNA bubble on negatively supercoiled DNA
Type: COMPLEX / Entity ID: #6-#7, #5, #1-#4 / Source: MULTIPLE SOURCES
Source (natural)Organism: Escherichia coli (E. coli)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 8
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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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: 2500 nm / Nominal defocus min: 800 nm
Image recordingElectron dose: 55.9 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.62 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 100010 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00534148
ELECTRON MICROSCOPYf_angle_d0.64946351
ELECTRON MICROSCOPYf_dihedral_angle_d14.75913077
ELECTRON MICROSCOPYf_chiral_restr0.0425267
ELECTRON MICROSCOPYf_plane_restr0.0045930

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