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- PDB-7zc4: Cryo-EM structure of POLRMT mutant. -

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Basic information

Entry
Database: PDB / ID: 7zc4
TitleCryo-EM structure of POLRMT mutant.
ComponentsDNA-directed RNA polymerase, mitochondrialPolymerase
KeywordsTRANSCRIPTION / Mitochondria / DNA dependent RNA polymerase.
Function / homology
Function and homology information


Mitochondrial transcription initiation / mitochondrial DNA-directed RNA polymerase complex / transcription initiation at mitochondrial promoter / mitochondrial promoter sequence-specific DNA binding / mitochondrial transcription / DNA primase activity / mitochondrial nucleoid / Transcriptional activation of mitochondrial biogenesis / DNA-directed 5'-3' RNA polymerase activity / DNA-directed RNA polymerase ...Mitochondrial transcription initiation / mitochondrial DNA-directed RNA polymerase complex / transcription initiation at mitochondrial promoter / mitochondrial promoter sequence-specific DNA binding / mitochondrial transcription / DNA primase activity / mitochondrial nucleoid / Transcriptional activation of mitochondrial biogenesis / DNA-directed 5'-3' RNA polymerase activity / DNA-directed RNA polymerase / 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)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.24 Å
AuthorsDas, H. / Hallberg, B.M.
Funding support1items
OrganizationGrant numberCountry
Not funded
CitationJournal: Cell / Year: 2022
Title: Non-coding 7S RNA inhibits transcription via mitochondrial RNA polymerase dimerization.
Authors: Xuefeng Zhu / Xie Xie / Hrishikesh Das / Benedict G Tan / Yonghong Shi / Ali Al-Behadili / Bradley Peter / Elisa Motori / Sebastian Valenzuela / Viktor Posse / Claes M Gustafsson / B Martin ...Authors: Xuefeng Zhu / Xie Xie / Hrishikesh Das / Benedict G Tan / Yonghong Shi / Ali Al-Behadili / Bradley Peter / Elisa Motori / Sebastian Valenzuela / Viktor Posse / Claes M Gustafsson / B Martin Hällberg / Maria Falkenberg /
Abstract: The mitochondrial genome encodes 13 components of the oxidative phosphorylation system, and altered mitochondrial transcription drives various human pathologies. A polyadenylated, non-coding RNA ...The mitochondrial genome encodes 13 components of the oxidative phosphorylation system, and altered mitochondrial transcription drives various human pathologies. A polyadenylated, non-coding RNA molecule known as 7S RNA is transcribed from a region immediately downstream of the light strand promoter in mammalian cells, and its levels change rapidly in response to physiological conditions. Here, we report that 7S RNA has a regulatory function, as it controls levels of mitochondrial transcription both in vitro and in cultured human cells. Using cryo-EM, we show that POLRMT dimerization is induced by interactions with 7S RNA. The resulting POLRMT dimer interface sequesters domains necessary for promoter recognition and unwinding, thereby preventing transcription initiation. We propose that the non-coding 7S RNA molecule is a component of a negative feedback loop that regulates mitochondrial transcription in mammalian cells.
History
DepositionMar 25, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0Feb 1, 2023Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: DNA-directed RNA polymerase, mitochondrial


Theoretical massNumber of molelcules
Total (without water)134,3281
Polymers134,3281
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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


Mass: 134327.703 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

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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: POLRMT / Type: ORGANELLE OR CELLULAR COMPONENT / Details: DNA-directed RNA polymerase, mitochondrial / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.137 MDa / Experimental value: YES
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
150 mMTrisTris1
2150 mMSodium ChlorideNaClSodium chloride1
35 mMMagnesium ChlorideMgCl21
SpecimenConc.: 2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
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
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 165000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 300 nm / Calibrated defocus min: 300 nm / Cs: 2.7 mm / C2 aperture diameter: 20 µm / Alignment procedure: ZEMLIN TABLEAU
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 1.5 sec. / Electron dose: 48.6 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 6583
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 10 eV

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Processing

SoftwareName: PHENIX / Version: 1.19_4085: / Classification: refinement
EM software
IDNameVersionCategory
1Warpparticle selection
2EPU2.7image acquisition
4cryoSPARC3.3.1CTF correction
7Coot0.9.3model fitting
10cryoSPARC3.3.1final Euler assignment
11cryoSPARC3.3.1classification
12cryoSPARC3.3.13D reconstruction
13PHENIX1.19model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.24 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 289680 / Symmetry type: POINT
Atomic model buildingProtocol: OTHER / Space: REAL
Atomic model buildingPDB-ID: 7ZPR

7zpr

Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0037448
ELECTRON MICROSCOPYf_angle_d0.66210129
ELECTRON MICROSCOPYf_dihedral_angle_d3.3031010
ELECTRON MICROSCOPYf_chiral_restr0.0431136
ELECTRON MICROSCOPYf_plane_restr0.0051316

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