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- PDB-9e88: De novo backtracked transcription elongation complex of Mycobacte... -

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Basic information

Entry
Database: PDB / ID: 9.0E+88
TitleDe novo backtracked transcription elongation complex of Mycobacterium tuberculosis RNA polymerase on a linear DNA fragment (TEC-Backtracked)
Components
  • (DNA-directed RNA polymerase subunit ...) x 4
  • DNA (27-MER)
  • DNA (33-MER)
  • RNA (5'-R(P*GP*CP*GP*AP*GP*AP*GP*GP*GP*AP*CP*AP*CP*G)-3')
  • Ubiquitin-like protein SMT3,RNA polymerase-binding transcription factor CarD
KeywordsTranscription/DNA/RNA / RNA polymerase / transcription / promoter escape / De novo / Transcription-DNA-RNA complex
Function / homology
Function and homology information


SUMO is conjugated to E1 (UBA2:SAE1) / SUMOylation of nuclear envelope proteins / SUMO is transferred from E1 to E2 (UBE2I, UBC9) / SUMO is proteolytically processed / SUMOylation of transcription factors / Postmitotic nuclear pore complex (NPC) reformation / SUMOylation of transcription cofactors / septin ring / SUMOylation of DNA damage response and repair proteins / Transcriptional and post-translational regulation of MITF-M expression and activity ...SUMO is conjugated to E1 (UBA2:SAE1) / SUMOylation of nuclear envelope proteins / SUMO is transferred from E1 to E2 (UBE2I, UBC9) / SUMO is proteolytically processed / SUMOylation of transcription factors / Postmitotic nuclear pore complex (NPC) reformation / SUMOylation of transcription cofactors / septin ring / SUMOylation of DNA damage response and repair proteins / Transcriptional and post-translational regulation of MITF-M expression and activity / SUMOylation of DNA replication proteins / SUMOylation of SUMOylation proteins / Recruitment and ATM-mediated phosphorylation of repair and signaling proteins at DNA double strand breaks / SUMOylation of RNA binding proteins / SUMOylation of chromatin organization proteins / ubiquitin-like protein ligase binding / rRNA transcription / protein sumoylation / DNA-directed RNA polymerase complex / condensed nuclear chromosome / ribonucleoside binding / protein tag activity / DNA-directed RNA polymerase / DNA-directed RNA polymerase activity / protein dimerization activity / DNA-templated transcription / magnesium ion binding / DNA binding / zinc ion binding / identical protein binding / nucleus / cytoplasm
Similarity search - Function
CarD-like, C-terminal domain / : / : / CarD, C-terminal domain / CarD-like/TRCF, RNAP-interacting domain / CarD-like/TRCF, RNAP-interacting domain superfamily / CarD-like/TRCF RID domain / CarD-like/TRCF domain / Rad60/SUMO-like domain / Ubiquitin-2 like Rad60 SUMO-like ...CarD-like, C-terminal domain / : / : / CarD, C-terminal domain / CarD-like/TRCF, RNAP-interacting domain / CarD-like/TRCF, RNAP-interacting domain superfamily / CarD-like/TRCF RID domain / CarD-like/TRCF domain / Rad60/SUMO-like domain / Ubiquitin-2 like Rad60 SUMO-like / 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 / 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, beta subunit, protrusion / RNA polymerase beta subunit / RNA polymerase Rpb1, domain 1 / 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, RBP11-like subunit / RNA polymerase Rpb1, funnel domain superfamily / RNA polymerase I subunit A N-terminus / 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 / Ubiquitin homologues / Ubiquitin domain profile. / Ubiquitin-like domain / Ubiquitin-like domain superfamily
Similarity search - Domain/homology
DNA / DNA (> 10) / DNA (> 100) / RNA / RNA (> 10) / DNA-directed RNA polymerase subunit omega / DNA-directed RNA polymerase subunit beta / DNA-directed RNA polymerase subunit alpha / DNA-directed RNA polymerase subunit beta' / RNA polymerase-binding transcription factor CarD / Ubiquitin-like protein SMT3
Similarity search - Component
Biological speciesMycobacterium tuberculosis (bacteria)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsBrewer, J.J. / Campbell, E.A. / Darst, S.A.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS) United States
CitationJournal: Nat Commun / Year: 2025
Title: Structural Insights into De Novo Promoter Escape by Mycobacterium tuberculosis RNA Polymerase.
Authors: Joshua Brewer / Madeleine Delbeau / Winston Bates Zoullas / Seth A Darst / Elizabeth A Campbell /
Abstract: Transcription in bacteria is a multi-step process. In the first step, contacts between RNA polymerase and the promoter DNA must be established for transcription initiation to begin, but then these ...Transcription in bacteria is a multi-step process. In the first step, contacts between RNA polymerase and the promoter DNA must be established for transcription initiation to begin, but then these contacts must be broken for the enzyme to transition into the elongation phase. Single-molecule and biochemical observations report that promoter escape is a highly regulated and sometimes rate-limiting step in the transcription cycle; however, the structural mechanisms of promoter escape remain obscure. Promoter escape also serves as the target for the clinically important antibiotic rifampicin, used to treat tuberculosis. Here, we present seven distinct intermediates showing the structural details of M. tuberculosis RNA polymerase initial transcribing complexes and promoter escape, using a de novo cryo-electron microscopy approach. We describe the structural rearrangements that RNA polymerase undergoes to clear the promoter, including those required to release the initiation factor, σ, providing a structural account for decades of biochemical observations. These structures and supporting biochemistry provide a model of promoter escape, a universal step in the transcription cycle, with conformations that may be used to develop Rifampicin alternatives.
History
DepositionNov 5, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Oct 1, 2025Provider: repository / Type: Initial release
Revision 1.0Oct 1, 2025Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Oct 1, 2025Data content type: Additional map / Part number: 1 / Data content type: Additional map / Provider: repository / Type: Initial release
Revision 1.0Oct 1, 2025Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Oct 1, 2025Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Oct 1, 2025Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Oct 1, 2025Data content type: Mask / Part number: 1 / Data content type: Mask / Provider: repository / Type: Initial release
Revision 1.0Oct 1, 2025Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release
Revision 1.1Dec 3, 2025Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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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
M: Ubiquitin-like protein SMT3,RNA polymerase-binding transcription factor CarD
N: DNA (27-MER)
R: RNA (5'-R(P*GP*CP*GP*AP*GP*AP*GP*GP*GP*AP*CP*AP*CP*G)-3')
T: DNA (33-MER)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)483,65512
Polymers483,5009
Non-polymers1553
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 ABCDE

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


Mass: 37745.328 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycobacterium tuberculosis (bacteria) / Gene: rpoA / Production host: Escherichia coli (E. coli) / References: UniProt: A5U8D3, DNA-directed RNA polymerase
#2: Protein DNA-directed RNA polymerase subunit beta / RNAP subunit beta / RNA polymerase subunit beta / Transcriptase subunit beta


Mass: 129905.672 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycobacterium tuberculosis (bacteria) / Gene: rpoB, MRA_0676 / Production host: Escherichia coli (E. coli) / References: UniProt: A5U052, DNA-directed RNA polymerase
#3: Protein DNA-directed RNA polymerase subunit beta' / RNAP subunit beta' / RNA polymerase subunit beta' / Transcriptase subunit beta'


Mass: 148882.969 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycobacterium tuberculosis (bacteria) / Gene: rpoC, BQ2027_MB0687 / Production host: Escherichia coli (E. coli) / References: UniProt: P0A675, DNA-directed RNA polymerase
#4: Protein DNA-directed RNA polymerase subunit omega / RNAP omega subunit / RNA polymerase omega subunit / Transcriptase subunit omega


Mass: 11851.140 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycobacterium tuberculosis (bacteria)
Gene: rpoZ, DKC2_1480, DSI35_24025, ERS007657_03145, ERS007661_02963, ERS007663_02972, ERS007665_03743, ERS007670_03615, ERS007679_02942, ERS007681_04445, ERS007722_03066, ERS007741_03196, ERS023446_ ...Gene: rpoZ, DKC2_1480, DSI35_24025, ERS007657_03145, ERS007661_02963, ERS007663_02972, ERS007665_03743, ERS007670_03615, ERS007679_02942, ERS007681_04445, ERS007722_03066, ERS007741_03196, ERS023446_03677, ERS024213_01369, ERS024276_01577, ERS027644_00478, ERS027646_01439, ERS027651_03169, ERS027653_00843, ERS027659_01429, ERS027661_02200, ERS027666_04715, ERS031537_03443, EU767_08910, EU768_15085, EU769_05250, EU770_14555, EU771_05130, EU773_14340, EU774_06465, EU775_07590, EU776_17830, EU777_06800, EUB02_12495, EUB03_09550, EUB06_03645, EUB07_12165, EUB08_05285, EUB09_00425, EUB10_04215, EUB11_10790, EUB13_01060, EUB14_01055, EUB16_00425, SAMEA2682864_01599, SAMEA2683035_01133
Production host: Escherichia coli (E. coli)
References: UniProt: A0A045H2R3, DNA-directed RNA polymerase

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DNA chain , 2 types, 2 molecules NT

#6: DNA chain DNA (27-MER)


Mass: 39318.250 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#8: DNA chain DNA (33-MER)


Mass: 39053.938 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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Protein / RNA chain , 2 types, 2 molecules MR

#5: Protein Ubiquitin-like protein SMT3,RNA polymerase-binding transcription factor CarD


Mass: 32104.199 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycobacterium tuberculosis (bacteria) / Gene: SMT3, YDR510W, D9719.15, carD, MT3689 / Production host: Escherichia coli (E. coli) / References: UniProt: Q12306, UniProt: P9WJG2
#7: RNA chain RNA (5'-R(P*GP*CP*GP*AP*GP*AP*GP*GP*GP*AP*CP*AP*CP*G)-3')


Mass: 6893.229 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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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
#10: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg

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Details

Has ligand of interestN
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: De novo backtracked transcription elongation complex of Mycobacterium tuberculosis RNA polymerase on a linear DNA fragment (TEC-Backtracked)
Type: COMPLEX / Entity ID: #1-#8 / Source: RECOMBINANT
Source (natural)Organism: Mycobacterium tuberculosis (bacteria)
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: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2200 nm / Nominal defocus min: 400 nm
Image recordingElectron dose: 51.83 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: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 40816 / Symmetry type: POINT

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