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- PDB-6c04: Mtb RNAP Holo/RbpA/double fork DNA -closed clamp -

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Basic information

Entry
Database: PDB / ID: 6c04
TitleMtb RNAP Holo/RbpA/double fork DNA -closed clamp
Components
  • (DNA-directed RNA polymerase subunit ...Polymerase) x 4
  • DNA (26-MER)
  • DNA (31-MER)
  • RNA polymerase sigma factor SigA
  • RNA polymerase-binding protein RbpA
Keywordstranscription/dna / initiation / transcription bubble / closed clamp / TRANSCRIPTION / transcription-dna 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 / peptidoglycan-based cell wall / DNA-directed RNA polymerase complex / 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 / peptidoglycan-based cell wall / DNA-directed RNA polymerase complex / 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 / Sigma-70 factors family signature 1. / RNA Polymerase Alpha Subunit; Chain A, domain 2 / RNA polymerase sigma factor RpoD, C-terminal / RNA polymerase sigma factor RpoD / RNA polymerase sigma-70 region 1.2 ...RNA polymerase-binding protein RbpA / RbpA superfamily / RNA polymerase-binding protein / DNA-directed RNA polymerase, insert domain / RNA polymerase, RBP11-like subunit / Sigma-70 factors family signature 1. / RNA Polymerase Alpha Subunit; Chain A, domain 2 / 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 / RNA polymerase Rpb1, clamp domain superfamily / RPB6/omega subunit-like superfamily / DNA-directed RNA polymerase, subunit beta-prime / RNA polymerase Rpb1, domain 3 / RNA polymerase Rpb1, domain 3 / RNA polymerase Rpb2, domain 2 superfamily / RNA polymerase Rpb1, domain 1 / RNA polymerase Rpb1, domain 1 / RNA polymerase, alpha subunit / RNA polymerase Rpb1, domain 4 / RNA polymerase Rpb1, domain 2 / RNA polymerase Rpb1, domain 4 / RNA polymerase, N-terminal / RNA polymerase Rpb1, funnel domain superfamily / RNA polymerase I subunit A N-terminus / RNA polymerase Rpb1, domain 5 / RNA polymerase Rpb1, domain 5 / RNA polymerase, beta subunit, protrusion / RNA polymerase beta subunit / 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
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 / 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: 3.27 Å
AuthorsDarst, S.A. / Campbell, E.A. / Boyaci Selcuk, H. / Chen, J. / Lilic, M.
Citation
Journal: 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.
#1: Journal: To Be Published
Title: Structure of Mycobacterium Tuberculosis RNAP Holo Enzyme/RbpA in closed clamp conformation
Authors: Darst, S.A. / Campbell, E.A. / Boyaci Selcuk, H. / Chen, J. / Lilic, M.
History
DepositionDec 27, 2017Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 28, 2018Provider: repository / Type: Initial release
Revision 1.1Dec 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 1.2Mar 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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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 (31-MER)
P: DNA (26-MER)
G: DNA (26-MER)
H: DNA (31-MER)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)471,85014
Polymers471,69511
Non-polymers1553
Water0
1


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


Mass: 130070.797 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' / Polymerase / RNAP subunit beta' / RNA polymerase subunit beta' / Transcriptase subunit beta'


Mass: 148202.219 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 / Polymerase / 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 / Production host: Escherichia coli (E. coli) / References: UniProt: A0A045IP01, UniProt: P9WHJ5*PLUS

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DNA chain , 2 types, 4 molecules OHPG

#7: DNA chain DNA (31-MER)


Mass: 9565.193 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) Mycobacterium tuberculosis (bacteria)
#8: DNA chain DNA (26-MER)


Mass: 7930.155 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) Mycobacterium tuberculosis (bacteria)

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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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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: Mtb RNAP Holo/RbpA/double fork DNA / 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: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy
Image recordingElectron dose: 6.7 e/Å2 / 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: 3.27 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 171547 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00728644
ELECTRON MICROSCOPYf_angle_d0.83739200
ELECTRON MICROSCOPYf_dihedral_angle_d16.99517171
ELECTRON MICROSCOPYf_chiral_restr0.0524465
ELECTRON MICROSCOPYf_plane_restr0.0074817

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