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- PDB-9gzm: Cryo-EM structure of the human mitochondrial RNA polymerase trans... -

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Basic information

Entry
Database: PDB / ID: 9gzm
TitleCryo-EM structure of the human mitochondrial RNA polymerase transcription initiation complex (POLRMT/TFAM/TFB2M/DNA/RNA) with a 2-mer RNA (pppGpA) and GTP poised for catalysis (pre-IC3)
Components
  • DNA-directed RNA polymerase, mitochondrial
  • Dimethyladenosine transferase 2, mitochondrial
  • Non-template strand DNA (56-MER)
  • RNA (5'-D(*(GTP))-R(P*A)-3')
  • Template strand DNA (56-MER)
  • Transcription factor A, mitochondrial
KeywordsTRANSCRIPTION / POLRMT / TFB2M / TFAM / transcription initiation / polymerase / h-mtRNAP / mtRNAP / RNA polymerase / mitochondrial DNA / mitochondria / slippage
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
GUANOSINE-5'-TRIPHOSPHATE / DNA / DNA (> 10) / RNA / 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: 3.4 Å
AuthorsGoovaerts, Q. / Shen, J. / Ajjugal, Y. / De Wijngaert, B. / Patel, S.S. / Das, K.
Funding support Belgium, 1items
OrganizationGrant numberCountry
Research Foundation - Flanders (FWO)1162823N Belgium
CitationJournal: Mol Cell / Year: 2025
Title: Human mitochondrial RNA polymerase structures reveal transcription start site and slippage mechanism.
Authors: Jiayu Shen / Quinten Goovaerts / Yogeeshwar Ajjugal / Brent De Wijngaert / Kalyan Das / Smita S Patel /
Abstract: Transcription of the human mitochondrial DNA is initiated by POLRMT and initiation factors mitochondrial transcription factor A (TFAM) and mitochondrial transcription factor B2 (TFB2M). We present ...Transcription of the human mitochondrial DNA is initiated by POLRMT and initiation factors mitochondrial transcription factor A (TFAM) and mitochondrial transcription factor B2 (TFB2M). We present cryo-electron microscopy (cryo-EM) structures of three transcription initiation intermediates (pre-catalytic IC3 [pre-IC3], slipped-IC3, and slipped pre-IC4) catalyzing RNA synthesis by normal and slippage pathways with fully resolved transcription bubbles and RNA transcripts starting from the +1 or -1 position. The structural and biochemical studies reveal mechanisms of promoter melting, start site selection, and slippage synthesis. Promoter melting begins at -4 with base-specific interactions of template -4 and -3 guanines with POLRMT and non-template -1 adenine with TFB2M. The NT-stabilizing loop (KLDPRSGGVIKPP) and Y209 of TFB2M and W1026 of POLRMT interact with the non-template strand to guide initiation from the +1 start site. The -1 position is not an alternative start site but supports slippage initiation by base-pairing with a slipped or rebound 2-nt RNA. Cryo-EM resolved additional apo and dimeric complexes whose populations may regulate transcription initiation.
History
DepositionOct 4, 2024Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jul 30, 2025Provider: repository / Type: Initial release
Revision 1.1Aug 6, 2025Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _citation_author.name / _em_admin.last_update
Revision 1.2Sep 3, 2025Group: Data collection / Database references / Category: citation / em_admin
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: DNA-directed RNA polymerase, mitochondrial
N: Non-template strand DNA (56-MER)
T: Template strand DNA (56-MER)
B: Dimethyladenosine transferase 2, mitochondrial
R: RNA (5'-D(*(GTP))-R(P*A)-3')
C: Transcription factor A, mitochondrial
hetero molecules


Theoretical massNumber of molelcules
Total (without water)237,0178
Polymers236,4706
Non-polymers5472
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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Protein , 3 types, 3 molecules ABC

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


Mass: 134632.344 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: POLRMT / Plasmid: pPROEXHTB / Production host: Escherichia coli BL21 (bacteria) / Variant (production host): CodonPlus RIL / References: UniProt: O00411, DNA-directed RNA polymerase
#4: 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: 38901.309 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: TFB2M, NS5ATP5 / Plasmid: pT7TEV-HMBP4 / Production host: Escherichia coli B (bacteria) / Variant (production host): ArcticExpress
References: UniProt: Q9H5Q4, Transferases; Transferring one-carbon groups; Methyltransferases
#6: 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: 27637.615 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: TFAM, TCF6, TCF6L2 / Plasmid: pPROEXHTB / Production host: Escherichia coli BL21 (bacteria) / Variant (production host): CodonPlus RIL / References: UniProt: Q00059

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DNA chain , 2 types, 2 molecules NT

#2: DNA chain Non-template strand DNA (56-MER)


Mass: 17486.254 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#3: DNA chain Template strand DNA (56-MER)


Mass: 17022.959 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

#5: RNA chain RNA (5'-D(*(GTP))-R(P*A)-3')


Mass: 789.413 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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Non-polymers , 2 types, 2 molecules

#7: Chemical ChemComp-GTP / GUANOSINE-5'-TRIPHOSPHATE


Mass: 523.180 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H16N5O14P3 / Comment: GTP, energy-carrying molecule*YM
#8: 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 interestN
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

Component
IDNameTypeEntity IDParent-IDSource
1Cryo-EM structure of the human mitochondrial RNA polymerase transcription initiation complex (POLRMT/TFAM/TFB2M/DNA/RNA) with a 2-mer RNA (pppGpA) and GTP poised for catalysis (pre-IC3)COMPLEX#1-#60RECOMBINANT
2DNA-directed RNA polymerase, mitochondrialORGANELLE OR CELLULAR COMPONENT#11RECOMBINANT
3DNA (56-mer)COMPLEX#2-#31RECOMBINANT
4Dimethyladenosine transferase 2, mitochondrialORGANELLE OR CELLULAR COMPONENT#41RECOMBINANT
5Transcription factor A, mitochondrialORGANELLE OR CELLULAR COMPONENT#61RECOMBINANT
Molecular weight
IDEntity assembly-IDValue (°)Experimental value
110.24 MDaYES
22
33
44
55
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
21Homo sapiens (human)9606
32Homo sapiens (human)9606
43Homo sapiens (human)9606
54Homo sapiens (human)9606
65Homo sapiens (human)9606
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
21Escherichia coli (E. coli)562
32Escherichia coli BL21 (bacteria)511693
43synthetic construct (others)32630
54Escherichia coli B (bacteria)37762
65Escherichia coli BL21 (bacteria)511693
Buffer solutionpH: 8
Details: 30 mM Tris-HCl pH 8, 200 mM NaCl, 3 mM DTT, 10 mM MgCl2
SpecimenConc.: 1.2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 K
Details: incubated on the grid for 5 seconds prior to double sided blotting for 3.5 seconds at force 1

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Electron microscopy imaging

MicroscopyModel: TFS GLACIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 130000 X / Nominal defocus max: 1800 nm / Nominal defocus min: 600 nm / Cs: 2.7 mm / C2 aperture diameter: 20 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: OTHER
Image recordingElectron dose: 40 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k) / Num. of grids imaged: 1
EM imaging opticsEnergyfilter name: TFS Selectris / Energyfilter slit width: 20 eV

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Processing

EM software
IDNameVersionCategory
1cryoSPARCparticle selection
2PHENIX1.19.2_4158:model refinement
13Coot3D reconstruction
CTF correctionType: NONE
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 175000 / Symmetry type: POINT
Atomic model buildingB value: 65 / Protocol: FLEXIBLE FIT / Space: REAL / Target criteria: Correlation Coefficient
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00315150
ELECTRON MICROSCOPYf_angle_d0.55720974
ELECTRON MICROSCOPYf_dihedral_angle_d18.5052747
ELECTRON MICROSCOPYf_chiral_restr0.0362332
ELECTRON MICROSCOPYf_plane_restr0.0052330

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