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- EMDB-16442: Cryo-EM structure of yeast mitochondrial RNA polymerase transcrip... -

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Entry
Database: EMDB / ID: EMD-16442
TitleCryo-EM structure of yeast mitochondrial RNA polymerase transcription initiation complex with 7-mer RNA, pppGpGpUpApApApU (IC7)
Map data
Sample
  • Complex: yeast mitochondrial RNA polymerase transcription initiation complex with 7-mer RNA, pppGpGpUpApApApu (IC7)
    • Complex: DNA-directed RNA polymerase, mitochondrial
      • Protein or peptide: DNA-directed RNA polymerase, mitochondrial
    • Complex: DNA (37-mer)
      • DNA: Non-Template DNA (37-MER)
      • DNA: Template DNA (37-MER)
    • Complex: Mitochondrial transcription factor 1
      • Protein or peptide: Mitochondrial transcription factor 1
    • Complex: RNA (pppGpGpUpApApApu)
      • RNA: RNA (pppGpGpUpApApApU)
  • Ligand: GUANOSINE-5'-TRIPHOSPHATE
KeywordsGene transcription / polymerase / RDRP / MTF1 / RPO41 / POLRMT / mtRNAP / DNA / transcription initiation / RNA polymerase / mitochondria / TRANSCRIPTION / IC7
Function / homology
Function and homology information


Mitochondrial transcription initiation / mitochondrial DNA-directed RNA polymerase complex / mitochondrial promoter sequence-specific DNA binding / mitochondrial transcription factor activity / transcription initiation at mitochondrial promoter / mitochondrial transcription / mitochondrial genome maintenance / DNA primase activity / DNA replication, synthesis of primer / positive regulation of DNA-templated transcription, elongation ...Mitochondrial transcription initiation / mitochondrial DNA-directed RNA polymerase complex / mitochondrial promoter sequence-specific DNA binding / mitochondrial transcription factor activity / transcription initiation at mitochondrial promoter / mitochondrial transcription / mitochondrial genome maintenance / DNA primase activity / DNA replication, synthesis of primer / positive regulation of DNA-templated transcription, elongation / mitochondrial nucleoid / Transferases; Transferring one-carbon groups; Methyltransferases / methyltransferase activity / mitochondrial intermembrane space / DNA-directed 5'-3' RNA polymerase activity / DNA-directed RNA polymerase / methylation / mitochondrial matrix / mitochondrion / DNA binding / RNA binding / cytoplasm
Similarity search - Function
Mitochondrial transcription factor Mtf1 / DNA-directed RNA polymerase, helix hairpin domain superfamily / 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 ...Mitochondrial transcription factor Mtf1 / DNA-directed RNA polymerase, helix hairpin domain superfamily / 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. / rRNA adenine dimethylase-like, C-terminal / Ribosomal RNA adenine methyltransferase KsgA/Erm / Ribosomal RNA adenine dimethylase / rRNA adenine N(6)-methyltransferase family profile. / S-adenosyl-L-methionine-dependent methyltransferase superfamily / DNA/RNA polymerase superfamily
Similarity search - Domain/homology
DNA-directed RNA polymerase, mitochondrial / Mitochondrial transcription factor 1
Similarity search - Component
Biological speciesSaccharomyces cerevisiae S288C (yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.75 Å
AuthorsGoovaerts Q / Shen J / Patel SS / Das K
Funding support Belgium, 1 items
OrganizationGrant numberCountry
KU Leuven Belgium
CitationJournal: Nature / Year: 2023
Title: Structures illustrate step-by-step mitochondrial transcription initiation.
Authors: Quinten Goovaerts / Jiayu Shen / Brent De Wijngaert / Urmimala Basu / Smita S Patel / Kalyan Das /
Abstract: Transcription initiation is a key regulatory step in gene expression during which RNA polymerase (RNAP) initiates RNA synthesis de novo, and the synthesized RNA at a specific length triggers the ...Transcription initiation is a key regulatory step in gene expression during which RNA polymerase (RNAP) initiates RNA synthesis de novo, and the synthesized RNA at a specific length triggers the transition to the elongation phase. Mitochondria recruit a single-subunit RNAP and one or two auxiliary factors to initiate transcription. Previous studies have revealed the molecular architectures of yeast and human mitochondrial RNAP initiation complexes (ICs). Here we provide a comprehensive, stepwise mechanism of transcription initiation by solving high-resolution cryogenic electron microscopy (cryo-EM) structures of yeast mitochondrial RNAP and the transcription factor Mtf1 catalysing two- to eight-nucleotide RNA synthesis at single-nucleotide addition steps. The growing RNA-DNA is accommodated in the polymerase cleft by template scrunching and non-template reorganization, creating stressed intermediates. During early initiation, non-template strand scrunching and unscrunching destabilize the short two- and three-nucleotide RNAs, triggering abortive synthesis. Subsequently, the non-template reorganizes into a base-stacked staircase-like structure supporting processive five- to eight-nucleotide RNA synthesis. The expanded non-template staircase and highly scrunched template in IC8 destabilize the promoter interactions with Mtf1 to facilitate initiation bubble collapse and promoter escape for the transition from initiation to the elongation complex (EC). The series of transcription initiation steps, each guided by the interplay of multiple structural components, reveal a finely tuned mechanism for potential regulatory control.
History
DepositionJan 10, 2023-
Header (metadata) releaseAug 30, 2023-
Map releaseAug 30, 2023-
UpdateNov 1, 2023-
Current statusNov 1, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_16442.png

Downloads & links

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Map

FileDownload / File: emd_16442.map.gz / Format: CCP4 / Size: 8.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
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AxesZ (Sec.)Y (Row.)X (Col.)
0.97 Å/pix.
x 130 pix.
= 126.1 Å		0.97 Å/pix.
x 130 pix.
= 126.1 Å		0.97 Å/pix.
x 130 pix.
= 126.1 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.97 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.018
Minimum - Maximum-0.17774852 - 0.26297197
Average (Standard dev.)0.00018450795 (±0.017546844)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions130130130
Spacing130130130
CellA=B=C: 126.100006 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_16442_msk_1.map
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Half map: #1

Fileemd_16442_half_map_1.map
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Half map: #2

Fileemd_16442_half_map_2.map
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Sample components

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Entire : yeast mitochondrial RNA polymerase transcription initiation compl...

EntireName: yeast mitochondrial RNA polymerase transcription initiation complex with 7-mer RNA, pppGpGpUpApApApu (IC7)
Components
  • Complex: yeast mitochondrial RNA polymerase transcription initiation complex with 7-mer RNA, pppGpGpUpApApApu (IC7)
    • Complex: DNA-directed RNA polymerase, mitochondrial
      • Protein or peptide: DNA-directed RNA polymerase, mitochondrial
    • Complex: DNA (37-mer)
      • DNA: Non-Template DNA (37-MER)
      • DNA: Template DNA (37-MER)
    • Complex: Mitochondrial transcription factor 1
      • Protein or peptide: Mitochondrial transcription factor 1
    • Complex: RNA (pppGpGpUpApApApu)
      • RNA: RNA (pppGpGpUpApApApU)
  • Ligand: GUANOSINE-5'-TRIPHOSPHATE

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Supramolecule #1: yeast mitochondrial RNA polymerase transcription initiation compl...

SupramoleculeName: yeast mitochondrial RNA polymerase transcription initiation complex with 7-mer RNA, pppGpGpUpApApApu (IC7)
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#5
Molecular weightTheoretical: 204 KDa

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Supramolecule #2: DNA-directed RNA polymerase, mitochondrial

SupramoleculeName: DNA-directed RNA polymerase, mitochondrial / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #2
Source (natural)Organism: Saccharomyces cerevisiae S288C (yeast)

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Supramolecule #3: DNA (37-mer)

SupramoleculeName: DNA (37-mer) / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #3-#4 / Details: 15S mitochondria
Source (natural)Organism: Saccharomyces cerevisiae S288C (yeast)

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Supramolecule #4: Mitochondrial transcription factor 1

SupramoleculeName: Mitochondrial transcription factor 1 / type: complex / ID: 4 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Saccharomyces cerevisiae S288C (yeast)

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Supramolecule #5: RNA (pppGpGpUpApApApu)

SupramoleculeName: RNA (pppGpGpUpApApApu) / type: complex / ID: 5 / Parent: 1 / Macromolecule list: #5
Source (natural)Organism: Saccharomyces cerevisiae S288C (yeast)

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Macromolecule #1: Mitochondrial transcription factor 1

MacromoleculeName: Mitochondrial transcription factor 1 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
EC number: Transferases; Transferring one-carbon groups; Methyltransferases
Source (natural)Organism: Saccharomyces cerevisiae S288C (yeast) / Strain: ATCC 204508 / S288C
Molecular weightTheoretical: 41.151203 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MGGSHHHHHH GMASSVPIPG IKDISKLKFF YGFKYLWNPT VYNKIFDKLD LTKTYKHPEE LKVLDLYPGV GIQSAIFYNK YCPRQYSLL EKRSSLYKFL NAKFEGSPLQ ILKRDPYDWS TYSNLIDEER IFVPEVQSSD HINDKFLTVA NVTGEGSEGL I MQWLSCIG ...String:
MGGSHHHHHH GMASSVPIPG IKDISKLKFF YGFKYLWNPT VYNKIFDKLD LTKTYKHPEE LKVLDLYPGV GIQSAIFYNK YCPRQYSLL EKRSSLYKFL NAKFEGSPLQ ILKRDPYDWS TYSNLIDEER IFVPEVQSSD HINDKFLTVA NVTGEGSEGL I MQWLSCIG NKNWLYRFGK VKMLLWMPST TARKLLARPG MHSRSKCSVV REAFTDTKLI AISDANELKG FDSQCIEEWD PI LFSAAEI WPTKGKPIAL VEMDPIDFDF DVDNWDYVTR HLMILKRTPL NTVMDSLGHG GQQYFNSRIT DKDLLKKCPI DLT NDEFIY LTKLFMEWPF KPDILMDFVD MYQTEHSG

UniProtKB: Mitochondrial transcription factor 1

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Macromolecule #2: DNA-directed RNA polymerase, mitochondrial

MacromoleculeName: DNA-directed RNA polymerase, mitochondrial / type: protein_or_peptide / ID: 2 / Details: Residues 101-1351 / Number of copies: 1 / Enantiomer: LEVO / EC number: DNA-directed RNA polymerase
Source (natural)Organism: Saccharomyces cerevisiae S288C (yeast) / Strain: ATCC 204508 / S288C
Molecular weightTheoretical: 143.282656 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: GAMGSGIQRP SAVTSMTRTR DVMQLWSLLE ACLQSNLMKR AFSILESLYL VPEHKQRFIE DYNMYLNSFS KNDPNFPILK MNEKLTNDL ETSFKDVNYN DKTLAIMIHH ALNFHSTTSS MLLKPIISAY LKMSVNGIRE IFSCLDILTI SDLNILMNDL K VITPSQLP ...String:
GAMGSGIQRP SAVTSMTRTR DVMQLWSLLE ACLQSNLMKR AFSILESLYL VPEHKQRFIE DYNMYLNSFS KNDPNFPILK MNEKLTNDL ETSFKDVNYN DKTLAIMIHH ALNFHSTTSS MLLKPIISAY LKMSVNGIRE IFSCLDILTI SDLNILMNDL K VITPSQLP NSVRPILESL TLSPTPVNNI ENEEGLNKVE AENDSKLHKA SNASSDSIKK PSLDPLREVS FHGSTEVLSK DA EKLIAVD TIGMRVIRHT LLGLSLTPEQ KEQISKFKFD ANDNVLKMKP TKNDDNNNSI NFFEIYNSLP TLEEKKAFES ALN IFNQDR QKVLENRATE AARERWKHDF EEAKARGDIS IEKNLNVKLW KWYNEMLPLV KEEINHCRSL LSEKLSDKKG LNKV DTNRL GYGPYLTLID PGKMCVITIL ELLKLNSTGG VIEGMRTARA VISVGKAIEM EFRSEQVLKS ESQAFRDVNK KSPEF KKLV QNAKSVFRSS QIEQSKILWP QSIRARIGSV LISMLIQVAK VSVQGVDPVT KAKVHGEAPA FAHGYQYHNG SKLGVL KIH KTLIRQLNGE RLIASVQPQL LPMLVEPKPW VNWRSGGYHY TQSTLLRTKD SPEQVAYLKA ASDNGDIDRV YDGLNVL GR TPWTVNRKVF DVVSQVWNKG EGFLDIPGAQ DEMVLPPAPP KNSDPSILRA WKLQVKTIAN KFSSDRSNRC DTNYKLEI A RAFLGEKLYF PHNLDFRGRA YPLSPHFNHL GNDMSRGLLI FWHGKKLGPS GLKWLKIHLS NLFGFDKLPL KDRVAFTES HLQDIKDSAE NPLTGDRWWT TADKPWQALA TCFELNEVMK MDNPEEFISH QPVHQDGTCN GLQHYAALGG DVEGATQVNL VPSDKPQDV YAHVARLVQK RLEIAAEKGD ENAKILKDKI TRKVVKQTVM TNVYGVTYVG ATFQIAKQLS PIFDDRKESL D FSKYLTKH VFSAIRELFH SAHLIQDWLG ESAKRISKSI RLDVDEKSFK NGNKPDFMSS VIWTTPLGLP IVQPYREESK KQ VETNLQT VFISDPFAVN PVNARRQKAG LPPNFIHSLD ASHMLLSAAE CGKQGLDFAS VHDSYWTHAS DIDTMNVVLR EQF IKLHEV DLVLRLKEEF DQRYKNYVKI GKLKRSTDLA QKIIRIRKDL SRKLGRSTTL ADEIYFEKKR QELLNSPLIE DRNV GEKMV TTVSLFEDIT DLDALELENG GDENSGMSVL LPLRLPEIPP KGDFDVTVLR NSQYFFS

UniProtKB: DNA-directed RNA polymerase, mitochondrial

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Macromolecule #3: Non-Template DNA (37-MER)

MacromoleculeName: Non-Template DNA (37-MER) / type: dna / ID: 3 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Saccharomyces cerevisiae S288C (yeast)
Molecular weightTheoretical: 11.468459 KDa
SequenceString:
(DC)(DG)(DA)(DA)(DT)(DA)(DA)(DG)(DT)(DA) (DT)(DT)(DG)(DA)(DT)(DA)(DT)(DA)(DA)(DG) (DT)(DA)(DA)(DT)(DA)(DA)(DA)(DT)(DG) (DC)(DA)(DA)(DA)(DT)(DT)(DG)(DC)

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Macromolecule #4: Template DNA (37-MER)

MacromoleculeName: Template DNA (37-MER) / type: dna / ID: 4 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Saccharomyces cerevisiae S288C (yeast)
Molecular weightTheoretical: 11.298308 KDa
SequenceString:
(DG)(DC)(DA)(DA)(DT)(DT)(DT)(DG)(DC)(DA) (DT)(DT)(DT)(DA)(DC)(DC)(DG)(DA)(DC)(DA) (DA)(DT)(DA)(DT)(DC)(DA)(DA)(DT)(DA) (DC)(DT)(DT)(DA)(DT)(DT)(DC)(DG)

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Macromolecule #5: RNA (pppGpGpUpApApApU)

MacromoleculeName: RNA (pppGpGpUpApApApU) / type: rna / ID: 5 / Number of copies: 1
Source (natural)Organism: Saccharomyces cerevisiae S288C (yeast)
Molecular weightTheoretical: 1.900197 KDa
SequenceString:
GUAAAU

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Macromolecule #6: GUANOSINE-5'-TRIPHOSPHATE

MacromoleculeName: GUANOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 6 / Number of copies: 1 / Formula: GTP
Molecular weightTheoretical: 523.18 Da
Chemical component information

ChemComp-GTP:
GUANOSINE-5'-TRIPHOSPHATE / GTP, energy-carrying molecule*YM

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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

Concentration0.7 mg/mL
BufferpH: 7
Details: 50 mM Bis-Tris propane; 100 mM NaCl, 5 mM MgCl2, 1 mM EDTA, 2 mM DTT
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 45 sec. / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.03 kPa
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 281.15 K / Instrument: LEICA EM GP / Details: 3 ul sample, back blotting for 12 seconds.

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Electron microscopy

MicroscopeTFS GLACIOS
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: COUNTING / Number grids imaged: 1 / Number real images: 1206 / Average exposure time: 37.06 sec. / Average electron dose: 40.0 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Calibrated defocus max: 2.6 µm / Calibrated defocus min: 0.55 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 1.8 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 150000
Sample stageSpecimen holder model: OTHER / Cooling holder cryogen: NITROGEN

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Image processing

Particle selectionNumber selected: 641911
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

6ymv

Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.75 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 96741
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final 3D classificationSoftware - Name: RELION (ver. 3.1)
FSC plot (resolution estimation)

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Atomic model buiding 1

DetailsRealspace refinement
RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Overall B value: 144 / Target criteria: correlation coefficient
Output model

PDB-8c5s:
Cryo-EM structure of yeast mitochondrial RNA polymerase transcription initiation complex with 7-mer RNA, pppGpGpUpApApApU (IC7)

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