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- EMDB-48417: Structure of the human mitochondrial promoter-initiated transcrip... -

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Basic information

Entry
Database: EMDB / ID: EMD-48417
TitleStructure of the human mitochondrial promoter-initiated transcription elongation complex, P-EC13
Map dataStructure of the human mitochondrial promoter-initiated transcription elongation complex, P-EC13
Sample
  • Complex: Structure of the human mitochondrial promoter-initiated transcription elongation complex, P-EC13
    • Protein or peptide: DNA-directed RNA polymerase, mitochondrial
    • DNA: Non-Template Strand DNA
    • RNA: RNA
    • DNA: Template Strand DNA
  • Ligand: 3'-DEOXY-CYTIDINE-5'-TRIPHOSPHATE
  • Ligand: MAGNESIUM ION
KeywordsMitochondrial RNA Polymerase / Transcription Initiation complex / TRANSCRIPTION / 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
DNA-directed RNA polymerase, mitochondrial
Similarity search - Component
Biological speciesHomo sapiens (human) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.74 Å
AuthorsHerbine KH / Nayak AR / Temiakov D
Funding support United States, 1 items
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, 2024-
Header (metadata) releaseAug 6, 2025-
Map releaseAug 6, 2025-
UpdateAug 6, 2025-
Current statusAug 6, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_48417.png

Downloads & links

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Map

FileDownload / File: emd_48417.map.gz / Format: CCP4 / Size: 216 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationStructure of the human mitochondrial promoter-initiated transcription elongation complex, P-EC13
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.73 Å/pix.
x 384 pix.
= 280.474 Å		0.73 Å/pix.
x 384 pix.
= 280.474 Å		0.73 Å/pix.
x 384 pix.
= 280.474 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.7304 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.1
Minimum - Maximum-0.03107896 - 1.9896839
Average (Standard dev.)0.0008899245 (±0.020665972)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions384384384
Spacing384384384
CellA=B=C: 280.4736 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: Unsharp Full map

Fileemd_48417_additional_1.map
AnnotationUnsharp Full map
Projections & Slices
AxesZYX

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Half map: Unsharp half-A map

Fileemd_48417_half_map_1.map
AnnotationUnsharp half-A map
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AxesZYX

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Density Histograms

Histogram

Histogram (log scale)

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Half map: Unsharp half-B map

Fileemd_48417_half_map_2.map
AnnotationUnsharp half-B map
Projections & Slices
AxesZYX

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Sample components

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Entire : Structure of the human mitochondrial promoter-initiated transcrip...

EntireName: Structure of the human mitochondrial promoter-initiated transcription elongation complex, P-EC13
Components
  • Complex: Structure of the human mitochondrial promoter-initiated transcription elongation complex, P-EC13
    • Protein or peptide: DNA-directed RNA polymerase, mitochondrial
    • DNA: Non-Template Strand DNA
    • RNA: RNA
    • DNA: Template Strand DNA
  • Ligand: 3'-DEOXY-CYTIDINE-5'-TRIPHOSPHATE
  • Ligand: MAGNESIUM ION

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Supramolecule #1: Structure of the human mitochondrial promoter-initiated transcrip...

SupramoleculeName: Structure of the human mitochondrial promoter-initiated transcription elongation complex, P-EC13
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4
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.
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 216 KDa

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Macromolecule #1: DNA-directed RNA polymerase, mitochondrial

MacromoleculeName: DNA-directed RNA polymerase, mitochondrial / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: DNA-directed RNA polymerase
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 138.818156 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MSALCWGRGA AGLKRALRPC GRPGLPGKEG TAGGVCGPRR SSSASPQEQD QDRRKDWGHV ELLEVLQARV RQLQAESVSE VVVNRVDVA RLPECGSGDG SLQPPRKVQM GAKDATPVPC GRWAKILEKD KRTQQMRMQR LKAKLQMPFQ SGEFKALTRR L QVEPRLLS ...String:
MSALCWGRGA AGLKRALRPC GRPGLPGKEG TAGGVCGPRR SSSASPQEQD QDRRKDWGHV ELLEVLQARV RQLQAESVSE VVVNRVDVA RLPECGSGDG SLQPPRKVQM GAKDATPVPC GRWAKILEKD KRTQQMRMQR LKAKLQMPFQ SGEFKALTRR L QVEPRLLS KQMAGCLEDC TRQAPESPWE EQLARLLQEA PGKLSLDVEQ APSGQHSQAQ LSGQQQRLLA FFKCCLLTDQ LP LAHHLLV VHHGQRQKRK LLTLDMYNAV MLGWARQGAF KELVYVLFMV KDAGLTPDLL SYAAALQCMG RQDQDAGTIE RCL EQMSQE GLKLQALFTA VLLSEEDRAT VLKAVHKVKP TFSLPPQLPP PVNTSKLLRD VYAKDGRVSY PKLHLPLKTL QCLF EKQLH MELASRVCVV SVEKPTLPSK EVKHARKTLK TLRDQWEKAL CRALRETKNR LEREVYEGRF SLYPFLCLLD EREVV RMLL QVLQALPAQG ESFTTLAREL SARTFSRHVV QRQRVSGQVQ ALQNHYRKYL CLLASDAEVP EPCLPRQYWE ELGAPE ALR EQPWPLPVQM ELGKLLAEML VQATQMPCSL DKPHRSSRLV PVLYHVYSFR NVQQIGILKP HPAYVQLLEK AAEPTLT FE AVDVPMLCPP LPWTSPHSGA FLLSPTKLMR TVEGATQHQE LLETCPPTAL HGALDALTQL GNCAWRVNGR VLDLVLQL F QAKGCPQLGV PAPPSEAPQP PEAHLPHSAA PARKAELRRE LAHCQKVARE MHSLRAEALY RLSLAQHLRD RVFWLPHNM DFRGRTYPCP PHFNHLGSDV ARALLEFAQG RPLGPHGLDW LKIHLVNLTG LKKREPLRKR LAFAEEVMDD ILDSADQPLT GRKWWMGAE EPWQTLACCM EVANAVRASD PAAYVSHLPV HQDGSCNGLQ HYAALGRDSV GAASVNLEPS DVPQDVYSGV A AQVEVFRR QDAQRGMRVA QVLEGFITRK VVKQTVMTVV YGVTRYGGRL QIEKRLRELS DFPQEFVWEA SHYLVRQVFK SL QEMFSGT RAIQHWLTES ARLISHMGSV VEWVTPLGVP VIQPYRLDSK VKQIGGGIQS ITYTHNGDIS RKPNTRKQKN GFP PNFIHS LDSSHMMLTA LHCYRKGLTF VSVHDCYWTH AADVSVMNQV CREQFVRLHS EPILQDLSRF LVKRFCSEPQ KILE ASQLK ETLQAVPKPG AFDLEQVKRS TYFFS

UniProtKB: DNA-directed RNA polymerase, mitochondrial

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Macromolecule #2: Non-Template Strand DNA

MacromoleculeName: Non-Template Strand DNA / type: dna / ID: 2
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.
Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 20.540199 KDa
SequenceString: (DG)(DT)(DG)(DT)(DT)(DA)(DG)(DT)(DT)(DA) (DG)(DG)(DG)(DA)(DG)(DT)(DG)(DA)(DC)(DT) (DG)(DT)(DT)(DA)(DA)(DA)(DA)(DG)(DT) (DG)(DC)(DA)(DT)(DA)(DC)(DC)(DG)(DC)(DC) (DA) (DA)(DG)(DA)(DG)(DA)(DA) ...String:
(DG)(DT)(DG)(DT)(DT)(DA)(DG)(DT)(DT)(DA) (DG)(DG)(DG)(DA)(DG)(DT)(DG)(DA)(DC)(DT) (DG)(DT)(DT)(DA)(DA)(DA)(DA)(DG)(DT) (DG)(DC)(DA)(DT)(DA)(DC)(DC)(DG)(DC)(DC) (DA) (DA)(DG)(DA)(DG)(DA)(DA)(DG)(DA) (DG)(DA)(DA)(DA)(DA)(DC)(DC)(DC)(DA)(DA) (DT)(DT) (DG)(DT)(DG)(DG)(DC)(DC)

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Macromolecule #4: Template Strand DNA

MacromoleculeName: Template Strand DNA / type: dna / ID: 4 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 20.162908 KDa
SequenceString: (DG)(DG)(DC)(DC)(DA)(DC)(DA)(DA)(DT)(DT) (DG)(DG)(DG)(DT)(DT)(DT)(DT)(DC)(DT)(DT) (DT)(DT)(DC)(DT)(DC)(DT)(DT)(DG)(DG) (DC)(DG)(DG)(DT)(DA)(DT)(DG)(DC)(DA)(DC) (DT) (DT)(DT)(DT)(DA)(DA)(DC) ...String:
(DG)(DG)(DC)(DC)(DA)(DC)(DA)(DA)(DT)(DT) (DG)(DG)(DG)(DT)(DT)(DT)(DT)(DC)(DT)(DT) (DT)(DT)(DC)(DT)(DC)(DT)(DT)(DG)(DG) (DC)(DG)(DG)(DT)(DA)(DT)(DG)(DC)(DA)(DC) (DT) (DT)(DT)(DT)(DA)(DA)(DC)(DA)(DG) (DT)(DC)(DA)(DC)(DT)(DC)(DC)(DC)(DT)(DA) (DA)(DC) (DT)(DA)(DA)(DC)(DA)(DC)

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Macromolecule #3: RNA

MacromoleculeName: RNA / type: rna / ID: 3 / Number of copies: 1
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 2.909871 KDa
SequenceString:
AAAGAAAAC

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Macromolecule #5: 3'-DEOXY-CYTIDINE-5'-TRIPHOSPHATE

MacromoleculeName: 3'-DEOXY-CYTIDINE-5'-TRIPHOSPHATE / type: ligand / ID: 5 / Number of copies: 1 / Formula: CH1
Molecular weightTheoretical: 467.157 Da
Chemical component information

ChemComp-CH1:
3'-DEOXY-CYTIDINE-5'-TRIPHOSPHATE

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Macromolecule #6: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 6 / Number of copies: 1 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.75 mg/mL
BufferpH: 7.9
Details: 20 mM Tris Buffer, pH 7.9, 150 mM NaCl, 10 mM DTT, and 10 mM MgCl2
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: GOLD / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 90 sec. / Pretreatment - Atmosphere: AIR / Details: 15 mA
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
Details5 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.

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Electron microscopy

MicroscopeTFS KRIOS
Specialist opticsEnergy filter - Name: TFS Selectris / Energy filter - Slit width: 10 eV
SoftwareName: Leginon
DetailsPreliminary grid screening performed manually using TFS Glacios.
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Number grids imaged: 1 / Number real images: 12245 / Average electron dose: 44.5 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.2 µm / Nominal defocus min: 0.3 µm / Nominal magnification: 165000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 5354890
Details: Automated particle picking (Blob picker, CryoSPARC) with manual inspection (Inspect Particle Picks).
CTF correctionSoftware - Name: cryoSPARC (ver. 4.6.0) / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: OTHER / Details: Ab-initio reconstruction in cryoSPARC
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 2.74 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.6.0) / Number images used: 43172
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.6.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.6.0)
Final 3D classificationNumber classes: 6 / Software - Name: cryoSPARC (ver. 4.6.0)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

6erp


Chain - Source name: PDB / Chain - Initial model type: experimental model
SoftwareName: Coot (ver. 0.9.8.2)
DetailsInitial docking of the starting model was done in Chimera and flexible/refined fitting done with Coot, and Phenix Real-Space Refinement.
RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Target criteria: Cross-correlation Coefficient
Output model

PDB-9mn9:
Structure of the human mitochondrial promoter-initiated transcription elongation complex, P-EC13

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