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- PDB-6z11: Structure of Mycobacterium smegmatis HelD protein in complex with... -

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Basic information

Entry
Database: PDB / ID: 6z11
TitleStructure of Mycobacterium smegmatis HelD protein in complex with RNA polymerase core - State III, primary channel dis-engaged and active site interfering
Components
  • (DNA-directed RNA polymerase subunit ...) x 4
  • RNA polymerase-associated transcription factor HelD
KeywordsTRANSCRIPTION / transcription cycle helicase-like protein RNA polymerase transcription
Function / homology
Function and homology information


DNA helicase complex / recombinational repair / 3'-5' DNA helicase activity / DNA-directed RNA polymerase complex / ribonucleoside binding / DNA-directed 5'-3' RNA polymerase activity / DNA-directed RNA polymerase / protein dimerization activity / hydrolase activity / response to antibiotic ...DNA helicase complex / recombinational repair / 3'-5' DNA helicase activity / DNA-directed RNA polymerase complex / ribonucleoside binding / DNA-directed 5'-3' RNA polymerase activity / DNA-directed RNA polymerase / protein dimerization activity / hydrolase activity / response to antibiotic / DNA-templated transcription / magnesium ion binding / DNA binding / zinc ion binding / ATP binding / cytosol / cytoplasm
Similarity search - Function
: / AAA domain / 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 / 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 ...: / AAA domain / 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 / 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 / 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 / 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, 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 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 / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
DNA-directed RNA polymerase subunit beta' / DNA-directed RNA polymerase subunit alpha / Superfamily protein I DNA or RNA helicase / DNA-directed RNA polymerase subunit omega / DNA-directed RNA polymerase subunit beta
Similarity search - Component
Biological speciesMycolicibacterium smegmatis MC2 155 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.36 Å
AuthorsKouba, T. / Koval, T. / Krasny, L. / Dohnalek, J.
CitationJournal: Nat Commun / Year: 2020
Title: Mycobacterial HelD is a nucleic acids-clearing factor for RNA polymerase.
Authors: Tomáš Kouba / Tomáš Koval' / Petra Sudzinová / Jiří Pospíšil / Barbora Brezovská / Jarmila Hnilicová / Hana Šanderová / Martina Janoušková / Michaela Šiková / Petr Halada / ...Authors: Tomáš Kouba / Tomáš Koval' / Petra Sudzinová / Jiří Pospíšil / Barbora Brezovská / Jarmila Hnilicová / Hana Šanderová / Martina Janoušková / Michaela Šiková / Petr Halada / Michal Sýkora / Ivan Barvík / Jiří Nováček / Mária Trundová / Jarmila Dušková / Tereza Skálová / URee Chon / Katsuhiko S Murakami / Jan Dohnálek / Libor Krásný /
Abstract: RNA synthesis is central to life, and RNA polymerase (RNAP) depends on accessory factors for recovery from stalled states and adaptation to environmental changes. Here, we investigated the mechanism ...RNA synthesis is central to life, and RNA polymerase (RNAP) depends on accessory factors for recovery from stalled states and adaptation to environmental changes. Here, we investigated the mechanism by which a helicase-like factor HelD recycles RNAP. We report a cryo-EM structure of a complex between the Mycobacterium smegmatis RNAP and HelD. The crescent-shaped HelD simultaneously penetrates deep into two RNAP channels that are responsible for nucleic acids binding and substrate delivery to the active site, thereby locking RNAP in an inactive state. We show that HelD prevents non-specific interactions between RNAP and DNA and dissociates stalled transcription elongation complexes. The liberated RNAP can either stay dormant, sequestered by HelD, or upon HelD release, restart transcription. Our results provide insights into the architecture and regulation of the highly medically-relevant mycobacterial transcription machinery and define HelD as a clearing factor that releases RNAP from nonfunctional complexes with nucleic acids.
History
DepositionMay 11, 2020Deposition site: PDBE / Processing site: PDBE
Revision 1.0Nov 4, 2020Provider: repository / Type: Initial release
Revision 1.1May 22, 2024Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_3d_fitting_list / pdbx_initial_refinement_model
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type

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Structure visualization

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  • Deposited structure unit
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  • EMDB-11026
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Structure viewerMolecule:
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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
H: RNA polymerase-associated transcription factor HelD
hetero molecules


Theoretical massNumber of molelcules
Total (without water)444,3109
Polymers444,1556
Non-polymers1553
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area38300 Å2
ΔGint-195 kcal/mol
Surface area131530 Å2
MethodPISA

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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)
Gene: rpoA, MSMEG_1524, MSMEI_1488 / 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)
Gene: rpoB, MSMEG_1367, MSMEI_1328 / Production host: Escherichia coli (E. coli) / References: UniProt: P60281, DNA-directed RNA polymerase
#3: 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)
Gene: rpoC, MSMEG_1368, MSMEI_1329 / Production host: Escherichia coli (E. coli) / References: UniProt: A0QS66, DNA-directed RNA polymerase
#4: 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 , 1 types, 1 molecules H

#5: Protein RNA polymerase-associated transcription factor HelD


Mass: 81298.078 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycolicibacterium smegmatis MC2 155 (bacteria)
Gene: MSMEG_2174 / Production host: Escherichia coli (E. coli) / References: UniProt: A0QUE0, DNA helicase

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Non-polymers , 2 types, 3 molecules

#6: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Zn
#7: 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

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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 smegmatis HelD protein in complex with RNA polymerase core
Type: COMPLEX / Entity ID: #1-#5 / Source: RECOMBINANT
Molecular weightValue: 0.444 MDa / Experimental value: NO
Source (natural)Organism: Mycolicibacterium smegmatis MC2 155 (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.8
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen 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 / Alignment procedure: ZEMLIN TABLEAU
Image recordingElectron dose: 50 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

EM software
IDNameVersionCategory
1Warpparticle selection
2EPUimage acquisition
4RELIONCTF correction
7Cootmodel fitting
9PHENIXmodel refinement
11RELIONfinal Euler assignment
12RELIONclassification
13RELION3.13D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.36 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 119100 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL
Atomic model building
IDPDB-ID 3D fitting-IDAccession codeInitial refinement model-IDSource nameType
16F6W

6f6w

16F6W1PDBexperimental model
26VSX

6vsx

16VSX2PDBexperimental model

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