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- PDB-9mn5: Structure of the human mitochondrial open transcription initiatio... -

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Basic information

Entry
Database: PDB / ID: 9mn5
TitleStructure of the human mitochondrial open transcription initiation complex, IC0
Components
  • DNA-directed RNA polymerase, mitochondrial
  • Dimethyladenosine transferase 2, mitochondrial
  • Non-Template strand
  • Template strand
  • Transcription factor A, mitochondrial
KeywordsTRANSCRIPTION / Mitochondrial RNA Polymerase / Transcription Initiation complex / POLRMT / TRANSCRIPTION-DNA-RNA complex
Function / homology
Function and homology information


rRNA (adenine-N6-)-methyltransferase activity / Mitochondrial transcription initiation / mitochondrial DNA-directed RNA polymerase complex / mitochondrial transcription factor activity / mitochondrial promoter sequence-specific DNA binding / mitochondrial respiratory chain complex assembly / transcription initiation at mitochondrial promoter / rRNA (adenine-N6,N6-)-dimethyltransferase activity / Strand-asynchronous mitochondrial DNA replication / mitochondrial DNA replication ...rRNA (adenine-N6-)-methyltransferase activity / Mitochondrial transcription initiation / mitochondrial DNA-directed RNA polymerase complex / mitochondrial transcription factor activity / mitochondrial promoter sequence-specific DNA binding / mitochondrial respiratory chain complex assembly / transcription initiation at mitochondrial promoter / rRNA (adenine-N6,N6-)-dimethyltransferase activity / Strand-asynchronous mitochondrial DNA replication / mitochondrial DNA replication / mitochondrial transcription / rRNA methylation / DNA binding, bending / mitochondrial nucleoid / heat shock protein binding / response to nutrient / Mitochondrial protein degradation / Transferases; Transferring one-carbon groups; Methyltransferases / Transcriptional activation of mitochondrial biogenesis / transcription coactivator binding / DNA-directed RNA polymerase / DNA-directed RNA polymerase activity / 3'-5'-RNA exonuclease activity / sequence-specific DNA binding / response to hypoxia / transcription cis-regulatory region binding / mitochondrial matrix / chromatin binding / regulation of transcription by RNA polymerase II / positive regulation of DNA-templated transcription / protein-containing complex / mitochondrion / RNA binding / nucleus / cytosol
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. ...: / 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. / Ribosomal RNA adenine methylase transferase, N-terminal / Ribosomal RNA adenine dimethylases / Ribosomal RNA adenine methyltransferase KsgA/Erm / Ribosomal RNA adenine dimethylase / rRNA adenine N(6)-methyltransferase family profile. / HMG-box domain / HMG (high mobility group) box / HMG boxes A and B DNA-binding domains profile. / high mobility group / High mobility group box domain / High mobility group box domain superfamily / Tetratricopeptide-like helical domain superfamily / S-adenosyl-L-methionine-dependent methyltransferase superfamily / DNA/RNA polymerase superfamily
Similarity search - Domain/homology
DNA / DNA (> 10) / DNA-directed RNA polymerase, mitochondrial / Transcription factor A, mitochondrial / Dimethyladenosine transferase 2, mitochondrial
Similarity search - Component
Biological speciesHomo sapiens (human)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.04 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
A: Transcription factor A, mitochondrial
B: Dimethyladenosine transferase 2, mitochondrial
E: DNA-directed RNA polymerase, mitochondrial
N: Non-Template strand
T: Template strand


Theoretical massNumber of molelcules
Total (without water)250,2895
Polymers250,2895
Non-polymers00
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

#1: Protein Transcription factor A, mitochondrial / mtTFA / Mitochondrial transcription factor 1 / MtTF1 / Transcription factor 6 / TCF-6 / ...mtTFA / Mitochondrial transcription factor 1 / MtTF1 / Transcription factor 6 / TCF-6 / Transcription factor 6-like 2


Mass: 29135.682 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: TFAM, TCF6, TCF6L2 / Production host: Escherichia coli (E. coli) / References: UniProt: Q00059
#2: Protein Dimethyladenosine transferase 2, mitochondrial / Hepatitis C virus NS5A-transactivated protein 5 / HCV NS5A-transactivated protein 5 / Mitochondrial ...Hepatitis C virus NS5A-transactivated protein 5 / HCV NS5A-transactivated protein 5 / Mitochondrial 12S rRNA dimethylase 2 / Mitochondrial transcription factor B2 / h-mtTFB / h-mtTFB2 / hTFB2M / mtTFB2 / S-adenosylmethionine-6-N' / N'-adenosyl(rRNA) dimethyltransferase 2


Mass: 45416.848 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: TFB2M, NS5ATP5 / Production host: Escherichia coli (E. coli)
References: UniProt: Q9H5Q4, Transferases; Transferring one-carbon groups; Methyltransferases
#3: 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
#4: DNA chain Non-Template strand


Mass: 18717.037 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#5: DNA chain Template strand


Mass: 18201.676 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
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 open transcription initiation complex, IC0
Type: COMPLEX
Details: Human Mitochondrial RNA polymerase (d119), TFAM (d42), and TFB2M (d20) assembled on promoter DNA containing a premelted bubble, and a 3-mer primer RNA.
Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.230 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: 20 uM mtRNAP (D119) was mixed with TFAM (D42), TFB2M (D20) and promoter DNA containing a premelted bubble with primer RNA (3-mer) at a 1:3:2:1 molar ratio in buffer containing 20 mM Tris- ...Details: 20 uM mtRNAP (D119) was mixed with TFAM (D42), TFB2M (D20) and promoter DNA containing a premelted bubble with primer RNA (3-mer) 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.
Specimen supportDetails: negatively glow-discharged with 15 mA for 80 seconds using a PELCO easiGlow Glow Discharge Cleaning System prior to Chameleon use
Grid material: GOLD / Grid type: SPT Labtech self-wicking R1.2/0.8
VitrificationInstrument: SPT LABTECH CHAMELEON / 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: 2500 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: 47.69 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k) / Num. of grids imaged: 2 / Num. of real images: 26965
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
9cryoSPARC4.6.0initial Euler assignment
10cryoSPARC4.6.0final Euler assignment
11cryoSPARC4.6.0classification
12cryoSPARC4.6.03D reconstruction
13PHENIX1.21rc1_5015model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 7863657
Details: Automated particle picking (Blob picker, CryoSPARC) with manual inspection (Inspect Particle Picks).
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.04 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 131267 / 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
RefinementHighest resolution: 3.04 Å
Stereochemistry target values: REAL-SPACE (WEIGHTED MAP SUM AT ATOM CENTERS)
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00415300
ELECTRON MICROSCOPYf_angle_d0.78321123
ELECTRON MICROSCOPYf_dihedral_angle_d22.6362719
ELECTRON MICROSCOPYf_chiral_restr0.0442329
ELECTRON MICROSCOPYf_plane_restr0.0072364

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