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- EMDB-14619: Cryo-EM structure of POLRMT mutant. -

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Basic information

Entry
Database: EMDB / ID: EMD-14619
TitleCryo-EM structure of POLRMT mutant.
Map data
Sample
  • Organelle or cellular component: POLRMT
    • Protein or peptide: DNA-directed RNA polymerase, mitochondrial
KeywordsMitochondria / DNA dependent RNA polymerase. / TRANSCRIPTION
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 / 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 / mitochondrial promoter sequence-specific DNA binding / transcription initiation at mitochondrial promoter / 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)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.24 Å
AuthorsDas H / Hallberg BM
Funding support1 items
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, 2022-
Header (metadata) releaseFeb 1, 2023-
Map releaseFeb 1, 2023-
UpdateJul 24, 2024-
Current statusJul 24, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_14619.png

Downloads & links

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Map

FileDownload / File: emd_14619.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.01 Å/pix.
x 300 pix.
= 303. Å		1.01 Å/pix.
x 300 pix.
= 303. Å		1.01 Å/pix.
x 300 pix.
= 303. Å

Surface

Projections

Slices (1/3)

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.01 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.07
Minimum - Maximum-0.031363502 - 2.208559
Average (Standard dev.)0.00053088024 (±0.015079622)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 303.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_14619_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_14619_half_map_2.map
Projections & Slices
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Histogram

Histogram (log scale)

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Sample components

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Entire : POLRMT

EntireName: POLRMT
Components
  • Organelle or cellular component: POLRMT
    • Protein or peptide: DNA-directed RNA polymerase, mitochondrial

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Supramolecule #1: POLRMT

SupramoleculeName: POLRMT / type: organelle_or_cellular_component / ID: 1 / Parent: 0 / Macromolecule list: all / Details: DNA-directed RNA polymerase, mitochondrial
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 137 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: 134.327703 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: QSSSASPQEQ DQDRRKDWGH VELLEVLQAR VRQLQAESVS EVVVNRVDVA RLPECGSGDG SLQPPRKVQM GAKDATPVPC GRWAKILEK DKRTQQMRMQ RLKAKLQMPF QSGEFKALTR RLQVEPRLLS KQMAGCLEDC TRQAPESPWE EQLARLLQEA P GKLSLDVE ...String:
QSSSASPQEQ DQDRRKDWGH VELLEVLQAR VRQLQAESVS EVVVNRVDVA RLPECGSGDG SLQPPRKVQM GAKDATPVPC GRWAKILEK DKRTQQMRMQ RLKAKLQMPF QSGEFKALTR RLQVEPRLLS KQMAGCLEDC TRQAPESPWE EQLARLLQEA P GKLSLDVE QAPSGQHSQA QLSGQQQRLL AFFKCCLLTD QLPLAHHLLV VHHGQAQKRA LLTLDMYNAV MLGWARQGAF KE LVYVLFM VKDAGLTPDL LSYAAALQCM GRQDQDAGTI ERCLEQMSQE GLKLQALFTA VLLSEEDRAT VLKAVHKVKP TFS LPPQLP PPVNTSKLLR DVYAKDGRVS YPKLHLPLKT LQCLFEKQLH MELASRVCVV SVEKPTLPSK EVKHARATLA TLRD QWEKA LCRALRETKN RLEREVYEGR FSLYPFLCLL DEREVVRMLL QVLQALPAQG ESFTTLAREL SARTFSRHVV QRQRV SGQV QALQNHYRKY LCLLASDAEV PEPCLPRQYW EELGAPEALR EQPWPLPVQM ELGKLLAEML VQATQMPCSL DKPHRS SRL VPVLYHVYSF RNVQQIGILK PHPAYVQLLE KAAEPTLTFE AVDVPMLCPP LPWTSPHSGA FLLSPTKLMR TVEGATQ HQ ELLETCPPTA LHGALDALTQ LGNCAWRVNG RVLDLVLQLF QAKGCPQLGV PAPPSEAPQP PEAHLPHSAA PARKAELR R ELAHCQKVAR EMHSLRAEAL YRLSLAQHLR DRVFWLPHNM DFRGRTYPCP PHFNHLGSDV ARALLEFAQG RPLGPHGLD WLKIHLVNLT GLKKREPLRK RLAFAEEVMD DILDSADQPL TGRKWWMGAE EPWQTLACCM EVANAVRASD PAAYVSHLPV HQDGSCNGL QHYAALGRDS VGAASVNLEP SDVPQDVYSG VAAQVEVFRR QDAQRGMRVA QVLEGFITRK VVKQTVMTVV Y GVTRYGGR LQIEKRLREL SDFPQEFVWE ASHYLVAQVF ASLQEMFSGT RAIQHWLTES ARLISHMGSV VEWVTPLGVP VI QPYRLDS KVKQIGGGIQ SITYTHNGDI SRAPNTAKQA NGFPPNFIHS LDSSHMMLTA LHCYRKGLTF VSVHDCYWTH AAD VSVMNQ VCREQFVRLH SEPILQDLSR FLVKRFCSEP QKILEASQLK ETLQAVPKPG AFDLEQVKRS TYFFS

UniProtKB: DNA-directed RNA polymerase, mitochondrial

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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

Concentration2 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
50.0 mMTrisTris
150.0 mMNaClSodium Chloride
5.0 mMMgCl2Magnesium Chloride
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS KRIOS
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 10 eV
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number grids imaged: 1 / Number real images: 6583 / Average exposure time: 1.5 sec. / Average electron dose: 48.6 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 20.0 µm / Calibrated defocus min: 0.3 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.5 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

Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

7pzr

Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.24 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.3.1) / Number images used: 289680
Initial angle assignmentType: PROJECTION MATCHING
Final angle assignmentType: PROJECTION MATCHING / Software - Name: cryoSPARC (ver. 3.3.1)
Final 3D classificationSoftware - Name: cryoSPARC (ver. 3.3.1)

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Atomic model buiding 1

Initial modelPDB ID:

7zpr


Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL / Protocol: OTHER
Output model

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

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