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- PDB-8r6p: Mycobacterium smegnatis RNA polymerase RP2-like transcription ini... -

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Basic information

Entry
Database: PDB / ID: 8r6p
TitleMycobacterium smegnatis RNA polymerase RP2-like transcription initiation complex with SigmaA, RbpA, HelD N-terminal domain and open promoter DNA
Components
  • (DNA 50-MER) x 2
  • (DNA-directed RNA polymerase subunit ...) x 4
  • Helicase
  • RNA polymerase sigma factor SigA
  • RNA polymerase-binding protein RbpA
KeywordsTRANSCRIPTION / HelD / RNA polymerase / transcription initiation / Mycobacteria / rifampicin resistance
Function / homology
Function and homology information


recombinational repair / 3'-5' DNA helicase activity / sigma factor activity / bacterial-type RNA polymerase core enzyme binding / DNA-directed RNA polymerase complex / DNA-templated transcription initiation / ribonucleoside binding / DNA-directed RNA polymerase / DNA-directed RNA polymerase activity / hydrolase activity ...recombinational repair / 3'-5' DNA helicase activity / sigma factor activity / bacterial-type RNA polymerase core enzyme binding / DNA-directed RNA polymerase complex / DNA-templated transcription initiation / ribonucleoside binding / DNA-directed RNA polymerase / DNA-directed RNA polymerase activity / hydrolase activity / protein dimerization activity / response to antibiotic / DNA-templated transcription / positive regulation of DNA-templated transcription / magnesium ion binding / DNA binding / zinc ion binding / ATP binding / cytoplasm / cytosol
Similarity search - Function
: / RNA polymerase-binding protein RbpA / RbpA superfamily / RNA polymerase-binding protein / UvrD/REP helicase N-terminal domain / UvrD-like DNA helicase ATP-binding domain profile. / DNA helicase, UvrD/REP type / UvrD-like helicase, ATP-binding domain / Sigma-70 factors family signature 1. / RNA polymerase sigma factor RpoD, C-terminal ...: / RNA polymerase-binding protein RbpA / RbpA superfamily / RNA polymerase-binding protein / UvrD/REP helicase N-terminal domain / UvrD-like DNA helicase ATP-binding domain profile. / DNA helicase, UvrD/REP type / UvrD-like helicase, ATP-binding domain / 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 / 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 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 Rpb2, domain 2 / RNA polymerase Rpb1, funnel domain superfamily / RNA polymerase Rpb2, domain 2 / 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 / Winged helix-like DNA-binding domain superfamily / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
DNA / DNA (> 10) / DNA-directed RNA polymerase subunit beta' / DNA-directed RNA polymerase subunit alpha / RNA polymerase sigma factor SigA / DNA-directed RNA polymerase subunit omega / RNA polymerase-binding protein RbpA / Helicase / DNA-directed RNA polymerase subunit beta
Similarity search - Component
Biological speciesMycolicibacterium smegmatis MC2 155 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.16 Å
AuthorsKoval, T. / Krasny, L. / Dohnalek, J. / Kouba, T.
Funding support Czech Republic, European Union, 5items
OrganizationGrant numberCountry
Grant Agency of the Czech Republic23-06295S Czech Republic
Ministry of Education (MoE, Czech Republic)LM2023042 Czech Republic
Czech Academy of Sciences86652036 Czech Republic
Grant Agency of the Czech Republic23-06295S Czech Republic
European Union (EU)LX22NPO5103European Union
CitationJournal: Nat Commun / Year: 2024
Title: Mycobacterial HelD connects RNA polymerase recycling with transcription initiation.
Authors: Tomáš Kovaľ / Nabajyoti Borah / Petra Sudzinová / Barbora Brezovská / Hana Šanderová / Viola Vaňková Hausnerová / Alena Křenková / Martin Hubálek / Mária Trundová / Kristýna ...Authors: Tomáš Kovaľ / Nabajyoti Borah / Petra Sudzinová / Barbora Brezovská / Hana Šanderová / Viola Vaňková Hausnerová / Alena Křenková / Martin Hubálek / Mária Trundová / Kristýna Adámková / Jarmila Dušková / Marek Schwarz / Jana Wiedermannová / Jan Dohnálek / Libor Krásný / Tomáš Kouba /
Abstract: Mycobacterial HelD is a transcription factor that recycles stalled RNAP by dissociating it from nucleic acids and, if present, from the antibiotic rifampicin. The rescued RNAP, however, must ...Mycobacterial HelD is a transcription factor that recycles stalled RNAP by dissociating it from nucleic acids and, if present, from the antibiotic rifampicin. The rescued RNAP, however, must disengage from HelD to participate in subsequent rounds of transcription. The mechanism of release is unknown. We show that HelD from Mycobacterium smegmatis forms a complex with RNAP associated with the primary sigma factor σ and transcription factor RbpA but not CarD. We solve several structures of RNAP-σ-RbpA-HelD without and with promoter DNA. These snapshots capture HelD during transcription initiation, describing mechanistic aspects of HelD release from RNAP and its protective effect against rifampicin. Biochemical evidence supports these findings, defines the role of ATP binding and hydrolysis by HelD in the process, and confirms the rifampicin-protective effect of HelD. Collectively, these results show that when HelD is present during transcription initiation, the process is protected from rifampicin until the last possible moment.
History
DepositionNov 22, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Oct 2, 2024Provider: repository / Type: Initial release
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Revision 1.1Dec 11, 2024Group: Data collection / Database references / Structure summary
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Revision 1.2Jul 9, 2025Group: Data collection / Category: em_admin / em_software / Item: _em_admin.last_update / _em_software.name
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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
F: RNA polymerase sigma factor SigA
H: Helicase
J: RNA polymerase-binding protein RbpA
D: DNA-directed RNA polymerase subunit beta'
E: DNA-directed RNA polymerase subunit omega
O: DNA 50-MER
P: DNA 50-MER
hetero molecules


Theoretical massNumber of molelcules
Total (without water)474,79513
Polymers474,64010
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: 37959.441 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycolicibacterium smegmatis MC2 155 (bacteria)
Production host: Escherichia coli (E. coli) / References: UniProt: A0QSL8, DNA-directed RNA polymerase
#2: Protein DNA-directed RNA polymerase subunit beta / RNAP subunit beta / RNA polymerase subunit beta / Transcriptase subunit beta


Mass: 128680.141 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycolicibacterium smegmatis MC2 155 (bacteria)
Production host: Escherichia coli (E. coli) / References: UniProt: P60281, DNA-directed RNA polymerase
#6: Protein DNA-directed RNA polymerase subunit beta' / RNAP subunit beta' / RNA polymerase subunit beta' / Transcriptase subunit beta'


Mass: 146712.891 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycolicibacterium smegmatis MC2 155 (bacteria)
Production host: Escherichia coli (E. coli) / References: UniProt: A0QS66
#7: Protein DNA-directed RNA polymerase subunit omega / RNAP omega subunit / RNA polymerase omega subunit / Transcriptase subunit omega


Mass: 11544.763 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycolicibacterium smegmatis MC2 155 (bacteria)
Gene: rpoZ, MSMEG_3053, MSMEI_2977 / Production host: Escherichia coli (E. coli) / References: UniProt: A0QWT1, DNA-directed RNA polymerase

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Protein , 3 types, 3 molecules FHJ

#3: Protein RNA polymerase sigma factor SigA


Mass: 51573.551 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycolicibacterium smegmatis MC2 155 (bacteria)
Gene: sigA, MSMEG_2758 / Production host: Escherichia coli (E. coli) / References: UniProt: A0QW02
#4: Protein Helicase


Mass: 16215.097 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycolicibacterium smegmatis MC2 155 (bacteria)
Gene: MSMEI_2121 / Production host: Escherichia coli (E. coli) / References: UniProt: I7G5V9
#5: Protein RNA polymerase-binding protein RbpA


Mass: 13078.731 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycolicibacterium smegmatis MC2 155 (bacteria)
Gene: rbpA, MSMEG_3858, MSMEI_3768 / Production host: Escherichia coli (E. coli) / References: UniProt: A0QZ11

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DNA chain , 2 types, 2 molecules OP

#8: DNA chain DNA 50-MER


Mass: 15521.958 Da / Num. of mol.: 1 / Source method: obtained synthetically
Source: (synth.) Mycolicibacterium smegmatis MC2 155 (bacteria)
#9: DNA chain DNA 50-MER


Mass: 15393.914 Da / Num. of mol.: 1 / Source method: obtained synthetically
Source: (synth.) Mycolicibacterium smegmatis MC2 155 (bacteria)

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Non-polymers , 2 types, 3 molecules

#10: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Zn
#11: 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: Mycobacterium smegnatis RNA polymerase RP2-like transcription initiation complex with SigmaA, RbpA, HelD N-terminal domain and open promoter DNA
Type: COMPLEX / Entity ID: #1-#9 / Source: MULTIPLE SOURCES
Source (natural)Organism: Mycolicibacterium smegmatis MC2 155 (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.4
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: 2900 nm / Nominal defocus min: 500 nm / Alignment procedure: ZEMLIN TABLEAU
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 2 sec. / Electron dose: 40 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)
Image scansMovie frames/image: 40

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Processing

EM software
IDNameVersionCategory
1Topazparticle selection
2SerialEMimage acquisition
9PHENIXmodel refinement
11RELION4final Euler assignment
13RELION43D reconstruction
CTF correctionType: PHASE FLIPPING ONLY
3D reconstructionResolution: 3.16 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 132995 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00229381
ELECTRON MICROSCOPYf_angle_d0.45940129
ELECTRON MICROSCOPYf_dihedral_angle_d14.3694640
ELECTRON MICROSCOPYf_chiral_restr0.0394552
ELECTRON MICROSCOPYf_plane_restr0.0034993

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