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- EMDB-40930: Reconstituted E. coli RNA polymerase post-termination complex on ... -

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Basic information

Entry
Database: EMDB / ID: EMD-40930
TitleReconstituted E. coli RNA polymerase post-termination complex on negatively-supercoiled DNA: closed duplex DNA (rPTCc)
Map dataLocally Filtered
Sample
  • Complex: E. coli Post-termination complex with open DNA bubble on negatively supercoiled DNA
    • Protein or peptide: DNA-directed RNA polymerase subunit alpha
    • Protein or peptide: DNA-directed RNA polymerase subunit beta
    • DNA: DNA (26-MER)
    • DNA: DNA (26-MER)
  • Protein or peptide: DNA-directed RNA polymerase subunit beta'
  • Ligand: MAGNESIUM ION
  • Ligand: ZINC ION
KeywordsRNA polymerase / Negatively supercoiled DNA / Sigma-independent transcription / TRANSCRIPTION
Function / homology
Function and homology information


DNA-directed RNA polymerase complex / ribonucleoside binding / : / : / : / : / : / : / DNA-directed RNA polymerase / protein dimerization activity ...DNA-directed RNA polymerase complex / ribonucleoside binding / : / : / : / : / : / : / DNA-directed RNA polymerase / protein dimerization activity / magnesium ion binding / DNA binding / zinc ion binding / cytoplasm
Similarity search - Function
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 / DNA-directed RNA polymerase, subunit beta-prime / RNA polymerase Rpb2, domain 2 superfamily ...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 / DNA-directed RNA polymerase, subunit beta-prime / RNA polymerase Rpb2, domain 2 superfamily / RNA polymerase Rpb1, domain 3 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 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 Rpb1, funnel domain superfamily / RNA polymerase I subunit A N-terminus / 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
DNA-directed RNA polymerase subunit beta' / DNA-directed RNA polymerase subunit alpha / DNA-directed RNA polymerase subunit beta
Similarity search - Component
Biological speciesEscherichia coli (E. coli) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.69 Å
AuthorsBrewer JJ / Darst SA / Campbell EA
Funding support United States, 1 items
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
DepositionMay 31, 2023-
Header (metadata) releaseApr 2, 2025-
Map releaseApr 2, 2025-
UpdateApr 2, 2025-
Current statusApr 2, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_40930.png

Downloads & links

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Map

FileDownload / File: emd_40930.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationLocally Filtered
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.08 Å/pix.
x 256 pix.
= 275.456 Å		1.08 Å/pix.
x 256 pix.
= 275.456 Å		1.08 Å/pix.
x 256 pix.
= 275.456 Å

Surface

Projections

Slices (1/3)

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.076 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.163
Minimum - Maximum-0.5009412 - 0.8791629
Average (Standard dev.)0.002606099 (±0.034752868)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 275.456 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: Sharpened

Fileemd_40930_additional_1.map
AnnotationSharpened
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half A

Fileemd_40930_half_map_1.map
AnnotationHalf A
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half B

Fileemd_40930_half_map_2.map
AnnotationHalf B
Projections & Slices
AxesZYX

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Histogram

Histogram (log scale)

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Sample components

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Entire : E. coli Post-termination complex with open DNA bubble on negative...

EntireName: E. coli Post-termination complex with open DNA bubble on negatively supercoiled DNA
Components
  • Complex: E. coli Post-termination complex with open DNA bubble on negatively supercoiled DNA
    • Protein or peptide: DNA-directed RNA polymerase subunit alpha
    • Protein or peptide: DNA-directed RNA polymerase subunit beta
    • DNA: DNA (26-MER)
    • DNA: DNA (26-MER)
  • Protein or peptide: DNA-directed RNA polymerase subunit beta'
  • Ligand: MAGNESIUM ION
  • Ligand: ZINC ION

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Supramolecule #1: E. coli Post-termination complex with open DNA bubble on negative...

SupramoleculeName: E. coli Post-termination complex with open DNA bubble on negatively supercoiled DNA
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2, #4-#5
Source (natural)Organism: Escherichia coli (E. coli)

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Macromolecule #1: DNA-directed RNA polymerase subunit alpha

MacromoleculeName: DNA-directed RNA polymerase subunit alpha / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO / EC number: DNA-directed RNA polymerase
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 25.897453 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: SVTEFLKPRL VDIEQVSSTH AKVTLEPLER GFGHTLGNAL RRILLSSMPG CAVTEVEIDG VLHEYSTKEG VQEDILEILL NLKGLAVRV QGKDEVILTL NKSGIGPVTA ADITHDGDVE IVKPQHVICH LTDENASISM RIKVQRGRGY VPASTRIHSE E DERPIGRL ...String:
SVTEFLKPRL VDIEQVSSTH AKVTLEPLER GFGHTLGNAL RRILLSSMPG CAVTEVEIDG VLHEYSTKEG VQEDILEILL NLKGLAVRV QGKDEVILTL NKSGIGPVTA ADITHDGDVE IVKPQHVICH LTDENASISM RIKVQRGRGY VPASTRIHSE E DERPIGRL LVDACYSPVE RIAYNVEAAR VEQRTDLDKL VIEMETNGTI DPEEAIRRAA TILAEQLEAF VDLRDV

UniProtKB: DNA-directed RNA polymerase subunit alpha

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Macromolecule #2: DNA-directed RNA polymerase subunit beta

MacromoleculeName: DNA-directed RNA polymerase subunit beta / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 150.560562 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: VYSYTEKKRI RKDFGKRPQV LDVPYLLSIQ LDSFQKFIEQ DPEGQYGLEA AFRSVFPIQS YSGNSELQYV SYRLGEPVFD VQECQIRGV TYSAPLRVKL RLVIYEREAP EGTVKDIKEQ EVYMGEIPLM TDNGTFVING TERVIVSQLH RSPGVFFDSD K GKTHSSGK ...String:
VYSYTEKKRI RKDFGKRPQV LDVPYLLSIQ LDSFQKFIEQ DPEGQYGLEA AFRSVFPIQS YSGNSELQYV SYRLGEPVFD VQECQIRGV TYSAPLRVKL RLVIYEREAP EGTVKDIKEQ EVYMGEIPLM TDNGTFVING TERVIVSQLH RSPGVFFDSD K GKTHSSGK VLYNARIIPY RGSWLDFEFD PKDNLFVRID RRRKLPATII LRALNYTTEQ ILDLFFEKVI FEIRDNKLQM EL VPERLRG ETASFDIEAN GKVYVEKGRR ITARHIRQLE KDDVKLIEVP VEYIAGKVVA KDYIDESTGE LICAANMELS LDL LAKLSQ SGHKRIETLF TNDLDHGPYI SETLRVDPTN DRLSALVEIY RMMRPGEPPT REAAESLFEN LFFSEDRYDL SAVG RMKFN RSLLREEIEG SGILSKDDII DVMKKLIDIR NGKGEVDDID HLGNRRIRSV GEMAENQFRV GLVRVERAVK ERLSL GDLD TLMPQDMINA KPISAAVKEF FGSSQLSQFM DQNNPLSEIT HKRRISALGP GGLTRERAGF EVRDVHPTHY GRVCPI ETP EGPNIGLINS LSVYAQTNEY GFLETPYRKV TDGVVTDEIH YLSAIEEGNY VIAQANSNLD EEGHFVEDLV TCRSKGE SS LFSRDQVDYM DVSTQQVVSV GASLIPFLEH DDANRALMGA NMQRQAVPTL RADKPLVGTG MERAVAVDSG VTAVAKRG G VVQYVDASRI VIKVNEDEMY PGEAGIDIYN LTKYTRSNQN TCINQMPCVS LGEPVERGDV LADGPSTDLG ELALGQNMR VAFMPWNGYN FEDSILVSER VVQEDRFTTI HIQELACVSR DTKLGPEEIT ADIPNVGEAA LSKLDESGIV YIGAEVTGGD ILVGKVTPK GETQLTPEEK LLRAIFGEKA SDVKDSSLRV PNGVSGTVID VQVFTRDGVE KDKRALEIEE MQLKQAKKDL S EELQILEA GLFSRIRAVL VAGGVEAEKL DKLPRDRWLE LGLTDEEKQN QLEQLAEQYD ELKHEFEKKL EAKRRKITQG DD LAPGVLK IVKVYLAVKR RIQPGDKMAG RHGNKGVISK INPIEDMPYD ENGTPVDIVL NPLGVPSRMN IGQILETHLG MAA KGIGDK INAMLKQQQE VAKLREFIQR AYDLGADVRQ KVDLSTFSDE EVMRLAENLR KGMPIATPVF DGAKEAEIKE LLKL GDLPT SGQIRLYDGR TGEQFERPVT VGYMYMLKLN HLVDDKMHAR STGSYSLVTQ QPLGGKAQFG GQRFGEMEVW ALEAY GAAY TLQEMLTVKS DDVNGRTKMY KNIVDGNHQM EPGMPESFNV LLKEIRSLGI NIELED

UniProtKB: DNA-directed RNA polymerase subunit beta

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Macromolecule #3: DNA-directed RNA polymerase subunit beta'

MacromoleculeName: DNA-directed RNA polymerase subunit beta' / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO / EC number: DNA-directed RNA polymerase
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 150.350234 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: EFDAIKIALA SPDMIRSWSF GEVKKPETIN YRTFKPERDG LFCARIFGPV KDYECLCGKY KRLKHRGVIC EKCGVEVTQT KVRRERMGH IELASPTAHI WFLKSLPSRI GLLLDMPLRD IERVLYFESY VVIEGGMTNL ERQQILTEEQ YLDALEEFGD E FDAKMGAE ...String:
EFDAIKIALA SPDMIRSWSF GEVKKPETIN YRTFKPERDG LFCARIFGPV KDYECLCGKY KRLKHRGVIC EKCGVEVTQT KVRRERMGH IELASPTAHI WFLKSLPSRI GLLLDMPLRD IERVLYFESY VVIEGGMTNL ERQQILTEEQ YLDALEEFGD E FDAKMGAE AIQALLKSMD LEQECEQLRE ELNETNSETK RKKLTKRIKL LEAFVQSGNK PEWMILTVLP VLPPDLRPLV PL DGGRFAT SDLNDLYRRV INRNNRLKRL LDLAAPDIIV RNEKRMLQEA VDALLDNGRR GRAITGSNKR PLKSLADMIK GKQ GRFRQN LLGKRVDYSG RSVITVGPYL RLHQCGLPKK MALELFKPFI YGKLELRGLA TTIKAAKKMV EREEAVVWDI LDEV IREHP VLLNRAPTLH RLGIQAFEPV LIEGKAIQLH PLVCAAYNAD FDGDQMAVHV PLTLEAQLEA RALMMSTNNI LSPAN GEPI IVPSQDVVLG LYYMTRDCVN AKGEGMVLTG PKEAERLYRS GLASLHARVK VRITEYEKDA NGELVAKTSL KDTTVG RAI LWMIVPKGLP YSIVNQALGK KAISKMLNTC YRILGLKPTV IFADQIMYTG FAYAARSGAS VGIDDMVIPE KKHEIIS EA EAEVAEIQEQ FQSGLVTAGE RYNKVIDIWA AANDRVSKAM MDNLQTETVI NRDGQEEKQV SFNSIYMMAD SGARGSAA Q IRQLAGMRGL MAKPDGSIIE TPITANFREG LNVLQYFIST HGARKGLADT ALKTANSGYL TRRLVDVAQD LVVTEDDCG THEGIMMTPV IEGGDVKEPL RDRVLGRVTA EDVLKPGTAD ILVPRNTLLH EQWCDLLEEN SVDAVKVRSV VSCDTDFGVC AHCYGRDLA RGHIINKGEA IGVIAAQSIG EPGTQLTMRT FHIGGAASRA AAESSIQVKN KGSIKLSNVK SVVNSSGKLV I TSRNTELK LIDEFGRTKE SYKVPYGAVL AKGDGEQVAG GETVANWDPH TMPVITEVSG FVRFTDMIDG QTITRQTDEL TG LSSLVVL DSAERTAGGK DLRPALKIVD AQGNDVLIPG TDMPAQYFLP GKAIVQLEDG VQISSGDTLA RIPQESGGTK DIT GGLPRV ADLFEARRPK EPAILAEISG IVSFGKETKG KRRLVITPVD GSDPYEEMIP KWRQLNVFEG ERVERGDVIS DGPE APHDI LRLRGVHAVT RYIVNEVQDV YRLQGVKIND KHIEVIVRQM LRKATIVNAG SSDFLEGEQV EYSRVKIANR ELEAN GKVG ATYSRDLLGI TKASLATESF ISAASFQETT RVLTEAAVAG KRDELRGLKE NVIVGRLIPA GTGYAYHQDA MRRR

UniProtKB: DNA-directed RNA polymerase subunit beta'

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Macromolecule #4: DNA (26-MER)

MacromoleculeName: DNA (26-MER) / type: dna / ID: 4 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 8.098421 KDa
SequenceString:
(DA)(DA)(DA)(DA)(DA)(DA)(DA)(DA)(DA)(DA) (DA)(DA)(DA)(DA)(DA)(DA)(DA)(DA)(DA)(DA) (DA)(DA)(DA)(DA)(DA)(DA)

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Macromolecule #5: DNA (26-MER)

MacromoleculeName: DNA (26-MER) / type: dna / ID: 5 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 7.864056 KDa
SequenceString:
(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT) (DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT) (DT)(DT)(DT)(DT)(DT)(DT)

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Macromolecule #6: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 6 / Number of copies: 1 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #7: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 7 / Number of copies: 2 / Formula: ZN
Molecular weightTheoretical: 65.409 Da

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 8
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 55.9 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.5 µm / Nominal defocus min: 0.8 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

6alh

Final reconstructionResolution.type: BY AUTHOR / Resolution: 4.69 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 13101
Initial angle assignmentType: PROJECTION MATCHING
Final angle assignmentType: PROJECTION MATCHING

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