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- EMDB-15556: Cryo-EM structure of yeast mitochondrial RNA polymerase transcrip... -

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Entry
Database: EMDB / ID: EMD-15556
TitleCryo-EM structure of yeast mitochondrial RNA polymerase transcription initiation complex with two GTP molecules poised for de novo initiation (IC2)
Map data
Sample
  • Complex: yeast mitochondrial RNA polymerase transcription initiation complex with two GTP molecules poised for de novo initiation (IC2)
    • Complex: DNA (33-mer)
      • DNA: Non-template DNA (33-MER)
      • DNA: Template DNA (33-MER)
    • Organelle or cellular component: DNA-directed RNA polymerase, mitochondrial
      • Protein or peptide: DNA-directed RNA polymerase, mitochondrial
    • Organelle or cellular component: Mitochondrial transcription factor 1
      • Protein or peptide: Mitochondrial transcription factor 1
  • Ligand: MAGNESIUM ION
  • Ligand: GUANOSINE-5'-TRIPHOSPHATE
Keywordsgene transcription / polymerase / RDRP / MTF1 / RPO41 / POLRMT / mtRNAP / DNA / transcription initiation / RNA polymerase / mitochondria / TRANSCRIPTION
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: 3.47 Å
AuthorsGoovaerts Q / Shen J / Patel SS / Das K
Funding support Belgium, 1 items
OrganizationGrant numberCountry
KU LeuvenKU Leuven start-up grant 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
DepositionAug 9, 2022-
Header (metadata) releaseAug 23, 2023-
Map releaseAug 23, 2023-
UpdateNov 1, 2023-
Current statusNov 1, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_15556.png

Downloads & links

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Map

FileDownload / File: emd_15556.map.gz / Format: CCP4 / Size: 15.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.76 Å/pix.
x 160 pix.
= 121.6 Å		0.76 Å/pix.
x 160 pix.
= 121.6 Å		0.76 Å/pix.
x 160 pix.
= 121.6 Å

Surface

Projections

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.76 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.016
Minimum - Maximum-0.14759718 - 0.24486186
Average (Standard dev.)0.00044655742 (±0.015562314)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions160160160
Spacing160160160
CellA=B=C: 121.6 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_15556_msk_1.map
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Half map: #2

Fileemd_15556_half_map_1.map
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Half map: #1

Fileemd_15556_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 two GTP molecules poised for de novo initiation (IC2)
Components
  • Complex: yeast mitochondrial RNA polymerase transcription initiation complex with two GTP molecules poised for de novo initiation (IC2)
    • Complex: DNA (33-mer)
      • DNA: Non-template DNA (33-MER)
      • DNA: Template DNA (33-MER)
    • Organelle or cellular component: DNA-directed RNA polymerase, mitochondrial
      • Protein or peptide: DNA-directed RNA polymerase, mitochondrial
    • Organelle or cellular component: Mitochondrial transcription factor 1
      • Protein or peptide: Mitochondrial transcription factor 1
  • Ligand: MAGNESIUM ION
  • 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 two GTP molecules poised for de novo initiation (IC2)
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4
Molecular weightTheoretical: 143 KDa

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Supramolecule #3: DNA (33-mer)

SupramoleculeName: DNA (33-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 #2: DNA-directed RNA polymerase, mitochondrial

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

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Supramolecule #4: Mitochondrial transcription factor 1

SupramoleculeName: Mitochondrial transcription factor 1 / type: organelle_or_cellular_component / ID: 4 / Parent: 1 / Macromolecule list: #1
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 (33-MER)

MacromoleculeName: Non-template DNA (33-MER) / type: dna / ID: 3 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Saccharomyces cerevisiae S288C (yeast)
Molecular weightTheoretical: 10.248671 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)(DG)(DA)(DT)(DA) (DA)(DT)(DG)(DC)

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Macromolecule #4: Template DNA (33-MER)

MacromoleculeName: Template DNA (33-MER) / type: dna / ID: 4 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Saccharomyces cerevisiae S288C (yeast)
Molecular weightTheoretical: 10.047511 KDa
SequenceString:
(DG)(DC)(DA)(DT)(DT)(DA)(DT)(DC)(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: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 5 / Number of copies: 1 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #6: GUANOSINE-5'-TRIPHOSPHATE

MacromoleculeName: GUANOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 6 / Number of copies: 2 / 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

Concentration0.8 mg/mL
BufferpH: 7
Details: 50mM Bis-tris propane, 100mM NaCl, 5mM MgCl2, 1mM EDTA, 2mM DTT
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / 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
Specialist opticsEnergy filter - Slit width: 20 eV
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: COUNTING / Number grids imaged: 1 / Number real images: 2045 / Average exposure time: 25.15 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: 2.2 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 190000
Sample stageSpecimen holder model: OTHER / Cooling holder cryogen: NITROGEN

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Image processing

Particle selectionNumber selected: 1149870
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

6ymv

Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.47 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 137631
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

Initial modelPDB ID:

6ymv


Chain - Source name: PDB / Chain - Initial model type: experimental model
DetailsRealspace refinement
RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Overall B value: 144 / Target criteria: Correlation coefficient
Output model

PDB-8ap1:
Cryo-EM structure of yeast mitochondrial RNA polymerase transcription initiation complex with two GTP molecules poised for de novo initiation (IC2)

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