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- PDB-6ymw: Cryo-EM structure of yeast mitochondrial RNA polymerase transcrip... -

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Basic information

Entry
Database: PDB / ID: 6ymw
TitleCryo-EM structure of yeast mitochondrial RNA polymerase transcription initiation complex
Components
  • Chains: N
  • Chains: T
  • DNA-directed RNA polymerase, mitochondrialPolymerase
  • Mitochondrial transcription factor 1
  • RNA (pppGpG)
KeywordsTRANSCRIPTION / gene transcription / polymerase / RDRP / MTF1 / RPO41 / POLRMT / mtRNAP / DNA / transcription initiation / RNA polymerase / mitochondria
Function / homology
Function and homology information


Mitochondrial transcription initiation / mitochondrial DNA-directed RNA polymerase complex / mitochondrial transcription factor activity / transcription initiation at mitochondrial promoter / mitochondrial promoter sequence-specific DNA binding / rRNA (adenine-N6,N6-)-dimethyltransferase activity / mitochondrial transcription / mitochondrial genome maintenance / DNA primase activity / DNA replication, synthesis of primer ...Mitochondrial transcription initiation / mitochondrial DNA-directed RNA polymerase complex / mitochondrial transcription factor activity / transcription initiation at mitochondrial promoter / mitochondrial promoter sequence-specific DNA binding / rRNA (adenine-N6,N6-)-dimethyltransferase activity / mitochondrial transcription / mitochondrial genome maintenance / DNA primase activity / DNA replication, synthesis of primer / positive regulation of DNA-templated transcription, elongation / mitochondrial nucleoid / Transferases; Transferring one-carbon groups; Methyltransferases / mitochondrial intermembrane space / DNA-directed 5'-3' RNA polymerase activity / DNA-directed RNA polymerase / mitochondrial matrix / mitochondrion / DNA binding / RNA binding / cytoplasm
Similarity search - Function
Mitochondrial transcription factor Mtf1 / DNA-directed RNA polymerase, helix hairpin domain superfamily / DNA-directed RNA polymerase, N-terminal / DNA-directed RNA polymerase, N-terminal domain superfamily / DNA-directed RNA polymerase N-terminal / Bacteriophage-type RNA polymerase family active site signature 1. / DNA-directed RNA polymerase N-terminal / DNA-directed RNA polymerase, phage-type / : / DNA-dependent RNA polymerase ...Mitochondrial transcription factor Mtf1 / DNA-directed RNA polymerase, helix hairpin domain superfamily / DNA-directed RNA polymerase, N-terminal / DNA-directed RNA polymerase, N-terminal domain superfamily / DNA-directed RNA polymerase N-terminal / Bacteriophage-type RNA polymerase family active site signature 1. / DNA-directed RNA polymerase N-terminal / DNA-directed RNA polymerase, phage-type / : / DNA-dependent RNA polymerase / Bacteriophage-type RNA polymerase family active site signature 2. / rRNA adenine dimethylase-like, C-terminal / Ribosomal RNA adenine methyltransferase KsgA/Erm / Ribosomal RNA adenine dimethylase / rRNA adenine N(6)-methyltransferase family profile. / S-adenosyl-L-methionine-dependent methyltransferase superfamily / DNA/RNA polymerase superfamily
Similarity search - Domain/homology
Chem-P5E / DNA / DNA (> 10) / RNA / DNA-directed RNA polymerase, mitochondrial / Mitochondrial transcription factor 1
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Saccharomyces cerevisiae S288C (yeast)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.71 Å
AuthorsDas, K. / Patel, S.S.
Funding support Belgium, 1items
OrganizationGrant numberCountry
KU LeuvenKU Leuven start-up grant Belgium
CitationJournal: Mol Cell / Year: 2021
Title: Cryo-EM Structures Reveal Transcription Initiation Steps by Yeast Mitochondrial RNA Polymerase.
Authors: Brent De Wijngaert / Shemaila Sultana / Anupam Singh / Chhaya Dharia / Hans Vanbuel / Jiayu Shen / Daniel Vasilchuk / Sergio E Martinez / Eaazhisai Kandiah / Smita S Patel / Kalyan Das /
Abstract: Mitochondrial RNA polymerase (mtRNAP) is crucial in cellular energy production, yet understanding of mitochondrial DNA transcription initiation lags that of bacterial and nuclear DNA transcription. ...Mitochondrial RNA polymerase (mtRNAP) is crucial in cellular energy production, yet understanding of mitochondrial DNA transcription initiation lags that of bacterial and nuclear DNA transcription. We report structures of two transcription initiation intermediate states of yeast mtRNAP that explain promoter melting, template alignment, DNA scrunching, abortive synthesis, and transition into elongation. In the partially melted initiation complex (PmIC), transcription factor MTF1 makes base-specific interactions with flipped non-template (NT) nucleotides "AAGT" at -4 to -1 positions of the DNA promoter. In the initiation complex (IC), the template in the expanded 7-mer bubble positions the RNA and NTP analog UTPαS, while NT scrunches into an NT loop. The scrunched NT loop is stabilized by the centrally positioned MTF1 C-tail. The IC and PmIC states coexist in solution, revealing a dynamic equilibrium between two functional states. Frequent scrunching/unscruching transitions and the imminent steric clashes of the inflating NT loop and growing RNA:DNA with the C-tail explain abortive synthesis and transition into elongation.
History
DepositionApr 10, 2020Deposition site: PDBE / Processing site: PDBE
Revision 1.0Dec 9, 2020Provider: repository / Type: Initial release
Revision 1.1Dec 16, 2020Group: Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_PubMed / _citation.title ..._citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _citation_author.name
Revision 1.2Feb 3, 2021Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.year
Revision 1.3Oct 18, 2023Group: 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_database_related / 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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Structure viewerMolecule:
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Assembly

Deposited unit
B: Mitochondrial transcription factor 1
A: DNA-directed RNA polymerase, mitochondrial
N: Chains: N
T: Chains: T
C: RNA (pppGpG)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)206,0607
Polymers205,5355
Non-polymers5252
Water0
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: light scattering, Dynamic Light Scattering
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area14100 Å2
ΔGint-70 kcal/mol
Surface area60590 Å2
MethodPISA

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Components

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Protein , 2 types, 2 molecules BA

#1: Protein Mitochondrial transcription factor 1 / Mitochondrial transcription factor mtTFB / Mitochondrial-specificity factor / RF1023


Mass: 41151.203 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / Gene: MTF1, YMR228W, YM9959.10 / Production host: Escherichia coli (E. coli)
References: UniProt: P14908, Transferases; Transferring one-carbon groups; Methyltransferases
#2: Protein DNA-directed RNA polymerase, mitochondrial / Polymerase


Mass: 143282.656 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / Gene: RPO41, YFL036W / Production host: Escherichia coli (E. coli) / References: UniProt: P13433, DNA-directed RNA polymerase

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DNA chain , 2 types, 2 molecules NT

#3: DNA chain Chains: N


Mass: 10248.671 Da / Num. of mol.: 1 / Source method: obtained synthetically / Details: 15S mitochondria / Source: (synth.) Saccharomyces cerevisiae S288C (yeast)
#4: DNA chain Chains: T


Mass: 10047.511 Da / Num. of mol.: 1 / Source method: obtained synthetically / Details: 15S mitochondria / Source: (synth.) Saccharomyces cerevisiae S288C (yeast)

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RNA chain , 1 types, 1 molecules C

#5: RNA chain RNA (pppGpG)


Mass: 805.413 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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Non-polymers , 2 types, 2 molecules

#6: Chemical ChemComp-P5E / [[(2~{R},3~{S},4~{R},5~{R})-5-[2,4-bis(oxidanylidene)pyrimidin-1-yl]-3,4-bis(oxidanyl)oxolan-2-yl]methoxy-sulfanyl-phosphoryl] phosphono hydrogen phosphate


Mass: 500.207 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C9H15N2O14P3S / Feature type: SUBJECT OF INVESTIGATION
#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 interestY

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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

Component
IDNameTypeEntity IDParent-IDSource
1mitochondria DNA-dependent RNA polymerase pre-initiation complexCOMPLEX#1-#50MULTIPLE SOURCES
2mitochondria DNA-dependent RNA polymerase pre-initiation complexCOMPLEX#1-#21RECOMBINANT
3DNACOMPLEX#3-#41RECOMBINANT
4RNA (pppGpG)COMPLEX#51RECOMBINANT
Molecular weightValue: 0.202 MDa / Experimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Saccharomyces cerevisiae (brewer's yeast)4932
33Saccharomyces cerevisiae (brewer's yeast)4932
44synthetic construct (others)32630
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
22Escherichia coli (E. coli)562
33synthetic construct (others)32630
44synthetic construct (others)32630
Buffer solutionpH: 7
Details: 50mM Bis-tris propane, 100mM NaCl, 5mM MgCl2, 1mM EDTA, 2mM DTT
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: LEICA EM GP / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 8 K / Details: 5 uL sample; back blotting for 12 -14 second

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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 / Nominal magnification: 165000 X / Nominal defocus max: 22000 nm / Nominal defocus min: 7000 nm / Calibrated defocus min: 5500 nm / Calibrated defocus max: 26000 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 6 sec. / Electron dose: 61 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 3500
EM imaging opticsEnergyfilter slit width: 20 eV
Image scansMovie frames/image: 50

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Processing

EM software
IDNameVersionCategory
1RELION3.08particle selection
2EPU2.6.1image acquisition
7Cootmodel fitting
9RELION3.08initial Euler assignment
10RELION3.08final Euler assignment
11RELION3.08classification
12RELION3.083D reconstruction
13PHENIX1.16model refinement
CTF correctionType: NONE
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.71 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 62807 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL / Target criteria: Coorelation coefficu=ient / Details: Realspace refinement
Atomic model building

3D fitting-ID: 1 / Source name: PDB / Type: experimental model

IDPDB-IDPdb chain-IDAccession codeInitial refinement model-ID
11I4W

1i4w

A1I4W1
26YMV

6ymv

6YMV2
RefinementStereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 55.32 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.007911731
ELECTRON MICROSCOPYf_angle_d1.055416141
ELECTRON MICROSCOPYf_chiral_restr0.07291773
ELECTRON MICROSCOPYf_plane_restr0.00491845
ELECTRON MICROSCOPYf_dihedral_angle_d20.31576826

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