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- PDB-9mn9: Structure of the human mitochondrial promoter-initiated transcrip... -

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Basic information

Entry
Database: PDB / ID: 9mn9
TitleStructure of the human mitochondrial promoter-initiated transcription elongation complex, P-EC13
Components
  • DNA-directed RNA polymerase, mitochondrial
  • Non-Template Strand DNA
  • RNA
  • Template Strand DNA
KeywordsTRANSCRIPTION / Mitochondrial RNA Polymerase / Transcription Initiation complex / POLRMT / TRANSCRIPTION-DNA-RNA complex
Function / homology
Function and homology information


Mitochondrial transcription initiation / mitochondrial DNA-directed RNA polymerase complex / mitochondrial promoter sequence-specific DNA binding / transcription initiation at mitochondrial promoter / Strand-asynchronous mitochondrial DNA replication / mitochondrial DNA replication / mitochondrial transcription / mitochondrial nucleoid / Transcriptional activation of mitochondrial biogenesis / DNA-directed RNA polymerase ...Mitochondrial transcription initiation / mitochondrial DNA-directed RNA polymerase complex / mitochondrial promoter sequence-specific DNA binding / transcription initiation at mitochondrial promoter / Strand-asynchronous mitochondrial DNA replication / mitochondrial DNA replication / mitochondrial transcription / mitochondrial nucleoid / Transcriptional activation of mitochondrial biogenesis / DNA-directed RNA polymerase / DNA-directed RNA polymerase activity / 3'-5'-RNA exonuclease activity / sequence-specific DNA binding / mitochondrial matrix / protein-containing complex / mitochondrion / RNA binding
Similarity search - Function
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. / Tetratricopeptide-like helical domain superfamily / DNA/RNA polymerase superfamily
Similarity search - Domain/homology
3'-DEOXY-CYTIDINE-5'-TRIPHOSPHATE / DNA / DNA (> 10) / RNA / DNA-directed RNA polymerase, mitochondrial
Similarity search - Component
Biological speciesHomo sapiens (human)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.74 Å
AuthorsHerbine, K.H. / Nayak, A.R. / Temiakov, D.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)NIH GM131832 United States
CitationJournal: Mol Cell / Year: 2025
Title: Structural basis for promoter recognition and transcription factor binding and release in human mitochondria.
Authors: Karl Herbine / Ashok R Nayak / Angelica Zamudio-Ochoa / Dmitry Temiakov /
Abstract: Transcription in human mitochondria is driven by a core apparatus consisting of a Pol A family RNA polymerase (mtRNAP), the initiation factors TFAM and TFB2M, and the elongation factor TEFM. While ...Transcription in human mitochondria is driven by a core apparatus consisting of a Pol A family RNA polymerase (mtRNAP), the initiation factors TFAM and TFB2M, and the elongation factor TEFM. While earlier structures of initiation and elongation complexes provided valuable snapshots, they represent isolated stages of a highly dynamic and multistep process. Critical aspects of mitochondrial transcription-such as DNA recognition and melting, promoter escape, and the release of initiation factors-remain poorly understood. Here, we present a series of cryoelectron microscopy (cryo-EM) structures that capture the transcription complex as it transitions from the initial open promoter complex to the processive elongation complex through intermediate stages. Our data reveal new, previously unidentified determinants of promoter specificity: the sequential disengagement of mtRNAP from TFAM and the promoter, the release of TFB2M, and the recruitment of TEFM. Together, these findings provide a detailed molecular mechanism underlying transcription in human mitochondria.
History
DepositionDec 20, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Aug 6, 2025Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
E: DNA-directed RNA polymerase, mitochondrial
N: Non-Template Strand DNA
R: RNA
T: Template Strand DNA
hetero molecules


Theoretical massNumber of molelcules
Total (without water)182,9236
Polymers182,4314
Non-polymers4912
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 chain , 2 types, 2 molecules NT

#2: DNA chain Non-Template Strand DNA


Mass: 20540.199 Da / Num. of mol.: 1 / Source method: obtained synthetically
Details: Chain break at GA, missing density for AAA at positions 46,47, and 48. This is not a mismatch! To submit we changed reference sequence from A to G to avoid this error, but please correct in final PDB validation.
Source: (synth.) synthetic construct (others)
#4: DNA chain Template Strand DNA


Mass: 20162.908 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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Protein / RNA chain , 2 types, 2 molecules ER

#1: Protein DNA-directed RNA polymerase, mitochondrial / MtRPOL


Mass: 138818.156 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: POLRMT / Production host: Escherichia coli (E. coli) / References: UniProt: O00411, DNA-directed RNA polymerase
#3: RNA chain RNA


Mass: 2909.871 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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Non-polymers , 2 types, 2 molecules

#5: Chemical ChemComp-CH1 / 3'-DEOXY-CYTIDINE-5'-TRIPHOSPHATE


Mass: 467.157 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C9H16N3O13P3 / Feature type: SUBJECT OF INVESTIGATION
#6: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION

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Details

Has ligand of interestY
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: Structure of the human mitochondrial promoter-initiated transcription elongation complex, P-EC13
Type: COMPLEX
Details: Human Mitochondrial RNA polymerase (d119), TFAM (d42), and TFB2M (d20) assembled on promoter DNA containing a premelted bubble, a 3-mer primer RNA, ATP, GTP, and 3'-deoxyCTP.
Entity ID: #1-#4 / Source: RECOMBINANT
Molecular weightValue: 0.216 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.9
Details: 20 mM Tris Buffer, pH 7.9, 150 mM NaCl, 10 mM DTT, and 10 mM MgCl2
SpecimenConc.: 0.75 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: 5 uM mtRNAP (D119) was mixed with TFAM (D42), TFB2M (D20) and promoter DNA at a 1:3:2:1 molar ratio in buffer containing 20 mM Tris-HCl, pH 7.9, 150 mM NaCl, 10 mM DTT, and 10 mM MgCl2 and ...Details: 5 uM mtRNAP (D119) was mixed with TFAM (D42), TFB2M (D20) and promoter DNA at a 1:3:2:1 molar ratio in buffer containing 20 mM Tris-HCl, pH 7.9, 150 mM NaCl, 10 mM DTT, and 10 mM MgCl2 and incubated for 10 minutes on ice prior to overnight dialysis at 4C in the same buffer. Following overnight dialysis, 5 uM IC was incubated with GAG RNA primer, ATP, GTP, and 3' deoxy CTP at a molar ratio of 1:10:25:25:50.
Specimen supportDetails: 15 mA / Grid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: UltrAuFoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Details: Preliminary grid screening performed manually using TFS Glacios.
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 165000 X / Nominal defocus max: 2200 nm / Nominal defocus min: 300 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 44.5 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 12245
EM imaging opticsEnergyfilter name: TFS Selectris / Energyfilter slit width: 10 eV

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Processing

EM software
IDNameVersionCategory
1cryoSPARC4.6.0particle selection
2Leginonimage acquisition
4cryoSPARC4.6.0CTF correction
7Coot0.9.8.2model fitting
9PHENIX1.21rc1_5015model refinement
10cryoSPARC4.6.0initial Euler assignment
11cryoSPARC4.6.0final Euler assignment
12cryoSPARC4.6.0classification
13cryoSPARC4.6.03D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 5354890
Details: Automated particle picking (Blob picker, CryoSPARC) with manual inspection (Inspect Particle Picks).
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.74 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 43172 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL / Target criteria: Cross-correlation Coefficient
Details: Initial docking of the starting model was done in Chimera and flexible/refined fitting done with Coot, and Phenix Real-Space Refinement.
Atomic model buildingPDB-ID: 6ERP

6erp


Accession code: 6ERP / Source name: PDB / Type: experimental model

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