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- PDB-9mn6: Structure of the human mitochondrial late-stage transcription ini... -

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Basic information

Entry
Database: PDB / ID: 9mn6
TitleStructure of the human mitochondrial late-stage transcription initiation complex, IC8
Components
  • DNA-directed RNA polymerase, mitochondrial
  • Dimethyladenosine transferase 2, 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


rRNA (adenine-N6-)-methyltransferase activity / Mitochondrial transcription initiation / mitochondrial DNA-directed RNA polymerase complex / mitochondrial transcription factor activity / mitochondrial promoter sequence-specific DNA binding / transcription initiation at mitochondrial promoter / rRNA (adenine-N6,N6-)-dimethyltransferase activity / Strand-asynchronous mitochondrial DNA replication / mitochondrial DNA replication / mitochondrial transcription ...rRNA (adenine-N6-)-methyltransferase activity / Mitochondrial transcription initiation / mitochondrial DNA-directed RNA polymerase complex / mitochondrial transcription factor activity / mitochondrial promoter sequence-specific DNA binding / transcription initiation at mitochondrial promoter / rRNA (adenine-N6,N6-)-dimethyltransferase activity / Strand-asynchronous mitochondrial DNA replication / mitochondrial DNA replication / mitochondrial transcription / rRNA methylation / mitochondrial nucleoid / Transferases; Transferring one-carbon groups; Methyltransferases / 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. / Ribosomal RNA adenine methylase transferase, N-terminal ...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. / Tetratricopeptide-like helical domain superfamily / S-adenosyl-L-methionine-dependent methyltransferase superfamily / DNA/RNA polymerase superfamily
Similarity search - Domain/homology
ADENOSINE-5'-TRIPHOSPHATE / DNA / DNA (> 10) / RNA / DNA-directed RNA polymerase, mitochondrial / Dimethyladenosine transferase 2, mitochondrial
Similarity search - Component
Biological speciesHomo sapiens (human)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.71 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

Downloads & links

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Assembly

Deposited unit
N: Non-Template Strand DNA
R: RNA
T: Template Strand DNA
E: DNA-directed RNA polymerase, mitochondrial
B: Dimethyladenosine transferase 2, mitochondrial
hetero molecules


Theoretical massNumber of molelcules
Total (without water)227,4167
Polymers226,8845
Non-polymers5312
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

#1: DNA chain Non-Template Strand DNA


Mass: 20524.199 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#3: 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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RNA chain , 1 types, 1 molecules R

#2: RNA chain RNA


Mass: 1962.277 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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Protein , 2 types, 2 molecules EB

#4: 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
#5: 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

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Non-polymers , 2 types, 2 molecules

#6: Chemical ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE


Mass: 507.181 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H16N5O13P3 / Feature type: SUBJECT OF INVESTIGATION / Comment: ATP, energy-carrying molecule*YM
#7: 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 late-stage transcription initiation complex, IC8
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, and 3'-deoxyGTP.
Entity ID: #1-#5 / 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, and 3' deoxy GTP at a molar ratio of 1:10:25:50.
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: 3200 nm / Nominal defocus min: 400 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: 45.44 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k) / Num. of grids imaged: 2 / Num. of real images: 17559
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: 2.71 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 147533 / 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: 2.71 Å
Stereochemistry target values: REAL-SPACE (WEIGHTED MAP SUM AT ATOM CENTERS)
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00512274
ELECTRON MICROSCOPYf_angle_d0.86216929
ELECTRON MICROSCOPYf_dihedral_angle_d19.4422093
ELECTRON MICROSCOPYf_chiral_restr0.0491882
ELECTRON MICROSCOPYf_plane_restr0.0091937

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