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- PDB-6bzo: Mtb RNAP Holo/RbpA/Fidaxomicin/upstream fork DNA -

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Basic information

Entry
Database: PDB / ID: 6bzo
TitleMtb RNAP Holo/RbpA/Fidaxomicin/upstream fork DNA
Components
  • (DNA-directed RNA polymerase subunit ...) x 4
  • DNA (26-MER)
  • DNA (32-MER)
  • RNA polymerase sigma factor SigA
  • RNA polymerase-binding protein RbpA
Keywordstranscription/dna/antibiotic / RNA Polymerase / Antibiotic / Inhibitor / TRANSCRIPTION / transcription-dna-antibiotic complex
Function / homology
Function and homology information


bacterial-type RNA polymerase holo enzyme binding / response to water / Antimicrobial action and antimicrobial resistance in Mtb / bacterial-type RNA polymerase core enzyme binding / sigma factor activity / DNA-directed RNA polymerase complex / peptidoglycan-based cell wall / DNA-templated transcription initiation / ribonucleoside binding / DNA-directed 5'-3' RNA polymerase activity ...bacterial-type RNA polymerase holo enzyme binding / response to water / Antimicrobial action and antimicrobial resistance in Mtb / bacterial-type RNA polymerase core enzyme binding / sigma factor activity / DNA-directed RNA polymerase complex / peptidoglycan-based cell wall / DNA-templated transcription initiation / ribonucleoside binding / DNA-directed 5'-3' RNA polymerase activity / DNA-directed RNA polymerase / nucleic acid binding / protein dimerization activity / response to antibiotic / DNA-templated transcription / positive regulation of DNA-templated transcription / magnesium ion binding / DNA binding / zinc ion binding / plasma membrane / cytosol / cytoplasm
Similarity search - Function
RNA polymerase-binding protein RbpA / RbpA superfamily / RNA polymerase-binding protein / DNA-directed RNA polymerase, insert domain / RNA polymerase, RBP11-like subunit / RNA Polymerase Alpha Subunit; Chain A, domain 2 / Sigma-70 factors family signature 1. / RNA polymerase sigma factor RpoD, C-terminal / RNA polymerase sigma factor RpoD / : ...RNA polymerase-binding protein RbpA / RbpA superfamily / RNA polymerase-binding protein / DNA-directed RNA polymerase, insert domain / RNA polymerase, RBP11-like subunit / RNA Polymerase Alpha Subunit; Chain A, domain 2 / Sigma-70 factors family signature 1. / RNA polymerase sigma factor RpoD, C-terminal / RNA polymerase sigma factor RpoD / : / RNA polymerase sigma-70 region 1.2 / Sigma-70 factor, region 1.2 / RNA polymerase sigma-70 region 3 / Sigma-70 region 3 / Sigma-70 factors family signature 2. / RNA polymerase sigma-70 / RNA polymerase sigma-70 region 4 / Sigma-70, region 4 / RNA polymerase sigma-70 region 2 / RNA polymerase sigma-70 like domain / Sigma-70 region 2 / RNA polymerase sigma factor, region 2 / RNA polymerase sigma factor, region 3/4-like / Gyrase A; domain 2 / 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 / Beta Complex / 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 / 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 / DNA-directed RNA polymerase, RpoA/D/Rpb3-type / RNA polymerase Rpb3/RpoA insert domain / RNA polymerase Rpb3/Rpb11 dimerisation domain / RNA polymerases D / 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 / Winged helix-like DNA-binding domain superfamily / 2-Layer Sandwich / Mainly Beta / Alpha Beta
Similarity search - Domain/homology
Fidaxomicin / DNA / DNA (> 10) / RNA polymerase sigma factor SigA / RNA polymerase-binding protein RbpA / DNA-directed RNA polymerase subunit alpha / DNA-directed RNA polymerase subunit beta' / DNA-directed RNA polymerase subunit omega / RNA polymerase sigma factor SigA / DNA-directed RNA polymerase subunit omega ...Fidaxomicin / DNA / DNA (> 10) / RNA polymerase sigma factor SigA / RNA polymerase-binding protein RbpA / DNA-directed RNA polymerase subunit alpha / DNA-directed RNA polymerase subunit beta' / DNA-directed RNA polymerase subunit omega / RNA polymerase sigma factor SigA / DNA-directed RNA polymerase subunit omega / DNA-directed RNA polymerase subunit beta' / DNA-directed RNA polymerase subunit beta / DNA-directed RNA polymerase subunit alpha / RNA polymerase-binding protein RbpA / DNA-directed RNA polymerase subunit beta
Similarity search - Component
Biological speciesMycobacterium tuberculosis (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.38 Å
AuthorsDarst, S.A. / Campbell, E.A. / Boyaci Selcuk, H. / Chen, J.
CitationJournal: Elife / Year: 2018
Title: Fidaxomicin jams RNA polymerase motions needed for initiation via RbpA contacts.
Authors: Hande Boyaci / James Chen / Mirjana Lilic / Margaret Palka / Rachel Anne Mooney / Robert Landick / Seth A Darst / Elizabeth A Campbell /
Abstract: Fidaxomicin (Fdx) is an antimicrobial RNA polymerase (RNAP) inhibitor highly effective against RNAP in vitro, but clinical use of Fdx is limited to treating intestinal infections due to poor ...Fidaxomicin (Fdx) is an antimicrobial RNA polymerase (RNAP) inhibitor highly effective against RNAP in vitro, but clinical use of Fdx is limited to treating intestinal infections due to poor absorption. To identify the structural determinants of Fdx binding to RNAP, we determined the 3.4 Å cryo-electron microscopy structure of a complete RNAP holoenzyme in complex with Fdx. We find that the actinobacteria general transcription factor RbpA contacts fidaxomycin, explaining its strong effect on . Additional structures define conformational states of RNAP between the free apo-holoenzyme and the promoter-engaged open complex ready for transcription. The results establish that Fdx acts like a doorstop to jam the enzyme in an open state, preventing the motions necessary to secure promoter DNA in the active site. Our results provide a structural platform to guide development of anti-tuberculosis antimicrobials based on the Fdx binding pocket.
History
DepositionDec 25, 2017Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 28, 2018Provider: repository / Type: Initial release
Revision 1.1Apr 24, 2019Group: Author supporting evidence / Data collection / Category: pdbx_audit_support
Revision 1.2Dec 18, 2019Group: Other / Category: atom_sites
Item: _atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] / _atom_sites.fract_transf_matrix[3][3]
Revision 1.3Mar 13, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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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 sigma factor SigA
J: RNA polymerase-binding protein RbpA
O: DNA (32-MER)
P: DNA (26-MER)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)455,41213
Polymers454,1999
Non-polymers1,2134
Water1086
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: mass spectrometry
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, rpoA_1, CLD25_18970, ERS007661_01801 / Production host: Escherichia coli (E. coli)
References: UniProt: A0A045J8T1, UniProt: P9WGZ1*PLUS, DNA-directed RNA polymerase
#2: Protein DNA-directed RNA polymerase subunit beta / RNAP subunit beta / RNA polymerase subunit beta / Transcriptase subunit beta


Mass: 130198.922 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycobacterium tuberculosis (bacteria) / Gene: rpoB, SAMEA2682864_01701 / Production host: Escherichia coli (E. coli)
References: UniProt: V9Z879, UniProt: P9WGY9*PLUS, DNA-directed RNA polymerase
#3: Protein DNA-directed RNA polymerase subunit beta' / RNAP subunit beta' / RNA polymerase subunit beta' / Transcriptase subunit beta'


Mass: 148074.094 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycobacterium tuberculosis (bacteria) / Gene: rpoC, rpoC_1, CLD25_03785 / Production host: Escherichia coli (E. coli)
References: UniProt: A0A045J9E2, UniProt: P9WGY7*PLUS, DNA-directed RNA polymerase
#4: Protein DNA-directed RNA polymerase subunit omega / RNAP omega subunit / RNA polymerase omega subunit / Transcriptase subunit omega


Mass: 11776.996 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycobacterium tuberculosis (bacteria) / Gene: rpoZ / Production host: Escherichia coli (E. coli)
References: UniProt: A0A0T9N9K3, UniProt: P9WGY5*PLUS, DNA-directed RNA polymerase

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Protein , 2 types, 2 molecules FJ

#5: Protein RNA polymerase sigma factor SigA


Mass: 58169.477 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycobacterium tuberculosis (bacteria) / Gene: sigA, hrdB_2, CLD25_14885 / Production host: Escherichia coli (E. coli) / References: UniProt: A0A045HD00, UniProt: P9WGI1*PLUS
#6: Protein RNA polymerase-binding protein RbpA


Mass: 12993.695 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycobacterium tuberculosis (bacteria) / Gene: rbpA, CLD25_11245 / Production host: Escherichia coli (E. coli) / References: UniProt: A0A045IP01, UniProt: P9WHJ5*PLUS

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DNA chain , 2 types, 2 molecules OP

#7: DNA chain DNA (32-MER)


Mass: 9565.193 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Mycobacterium tuberculosis (bacteria)
#8: DNA chain DNA (26-MER)


Mass: 7930.155 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Mycobacterium tuberculosis (bacteria)

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Non-polymers , 4 types, 10 molecules

#9: Chemical ChemComp-FI8 / Fidaxomicin


Mass: 1058.039 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C52H74Cl2O18 / Feature type: SUBJECT OF INVESTIGATION / Comment: antibiotic*YM
#10: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Formula: Zn
#11: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Formula: Mg
#12: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 6 / Source method: isolated from a natural source / Formula: H2O

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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: Complex of Mtb RNAP with an antibiotic called fidaxomicin
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
Specimen supportGrid material: GOLD / Grid mesh size: 400 divisions/in. / Grid type: C-flat
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 %

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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: 1800 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm
Specimen holderSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 6.7 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.12_2829: / Classification: refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.38 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 173509 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00227904
ELECTRON MICROSCOPYf_angle_d0.49138109
ELECTRON MICROSCOPYf_dihedral_angle_d13.90116814
ELECTRON MICROSCOPYf_chiral_restr0.0384325
ELECTRON MICROSCOPYf_plane_restr0.0044794

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