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- PDB-8q63: Cryo-EM structure of IC8', a second state of yeast mitochondrial ... -

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Basic information

Entry
Database: PDB / ID: 8q63
TitleCryo-EM structure of IC8', a second state of yeast mitochondrial RNA polymerase transcription initiation complex with 8-mer RNA, pppGpGpUpApApApUpG
Components
  • DNA-directed RNA polymerase, mitochondrial
  • Mitochondrial transcription factor 1
  • Non-template DNA (37-mer)
  • RNA (pppGpGpUpApApApUpG)
  • Template DNA (37-MER)
KeywordsTRANSCRIPTION / gene transcription / polymerase / RDRP / MTF1 / RPO41 / mtRNAP / DNA / transcription initiation / RNA polymerase / mitochondria / IC8' / IC8
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
GUANOSINE-5'-TRIPHOSPHATE / DNA / DNA (> 10) / RNA / DNA-directed RNA polymerase, mitochondrial / Mitochondrial transcription factor 1
Similarity search - Component
Biological speciesSaccharomyces cerevisiae S288C (yeast)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.68 Å
AuthorsGoovaerts, Q. / Shen, J. / Patel, S.S. / Das, K.
Funding support Belgium, 1items
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
DepositionAug 10, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Aug 30, 2023Provider: repository / Type: Initial release
Revision 1.1Oct 11, 2023Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.title / _citation.year
Revision 1.2Oct 25, 2023Group: Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID
Revision 1.3Nov 1, 2023Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
B: Mitochondrial transcription factor 1
A: DNA-directed RNA polymerase, mitochondrial
N: Non-template DNA (37-mer)
T: Template DNA (37-MER)
C: RNA (pppGpGpUpApApApUpG)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)209,9696
Polymers209,4465
Non-polymers5231
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable, gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

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Protein , 2 types, 2 molecules BA

#1: Protein Mitochondrial transcription factor 1 / Mitochondrial transcription factor mtTFB / Mitochondrial-specificity factor / RF1023


Mass: 41151.203 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae S288C (yeast) / Strain: ATCC 204508 / S288C / Gene: MTF1, YMR228W, YM9959.10 / Plasmid: pTrcHisC / Production host: Escherichia coli BL21(DE3) (bacteria)
References: UniProt: P14908, Transferases; Transferring one-carbon groups; Methyltransferases
#2: Protein DNA-directed RNA polymerase, mitochondrial


Mass: 143282.656 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae S288C (yeast) / Strain: ATCC 204508 / S288c / Gene: RPO41, YFL036W / Plasmid: ProEXHTB / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): CodonPlus RIL / References: UniProt: P13433, DNA-directed RNA polymerase

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DNA chain , 2 types, 2 molecules NT

#3: DNA chain Non-template DNA (37-mer)


Mass: 11468.459 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Saccharomyces cerevisiae S288C (yeast)
#4: DNA chain Template DNA (37-MER)


Mass: 11298.308 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Saccharomyces cerevisiae S288C (yeast)

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RNA chain / Non-polymers , 2 types, 2 molecules C

#5: RNA chain RNA (pppGpGpUpApApApUpG)


Mass: 2245.403 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Saccharomyces cerevisiae S288C (yeast)
#6: 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

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Details

Has ligand of interestN

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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-IDSourceDetails
1alternate state of yeast mitochondrial RNA polymerase transcription initiation complex with 8-mer RNA, pppGpGpUpApApApUpG, and a wider polymerase cleft (IC8')COMPLEX#1-#40MULTIPLE SOURCES
2DNA-directed RNA polymerase, mitochondrialCOMPLEX#21RECOMBINANT
3Mitochondrial transcription factor 1COMPLEX#11RECOMBINANT
4DNA (37-mer)COMPLEX#3-#41RECOMBINANT15S mitochondria
5RNA (pppGpGpUpApApApUpG)COMPLEX#51RECOMBINANT
Molecular weight
IDEntity assembly-IDValue (°)Experimental value
110.204 MDaNO
22
33
44
55
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
21Saccharomyces cerevisiae S288C (yeast)559292
32Saccharomyces cerevisiae S288C (yeast)559292
43Saccharomyces cerevisiae S288C (yeast)559292
54Saccharomyces cerevisiae S288C (yeast)559292
65Saccharomyces cerevisiae S288C (yeast)559292
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
21Escherichia coli BL21(DE3) (bacteria)469008
32Escherichia coli BL21(DE3) (bacteria)469008
43Escherichia coli BL21(DE3) (bacteria)469008
54synthetic construct (others)32630
65synthetic construct (others)32630
Buffer solutionpH: 7
Details: 50 mM Bis-Tris propane; 100 mM NaCl, 5 mM MgCl2, 1 mM EDTA, 2 mM DTT
SpecimenConc.: 0.5 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: LEICA EM GP / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 281.15 K / Details: 3 ul sample, back blotting for 12 seconds

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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: 150000 X / Nominal defocus max: 1800 nm / Nominal defocus min: 800 nm / Calibrated defocus min: 550 nm / Calibrated defocus max: 2600 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: OTHER
Image recordingAverage exposure time: 37.06 sec. / Electron dose: 40 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON III (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 1409

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Processing

EM software
IDNameVersionCategory
1RELION3.1particle selection
2EPU2.9.0image acquisition
4CTFFIND4CTF correction
7Cootmodel fitting
9RELION3.1initial Euler assignment
10RELION3.1final Euler assignment
11RELION3.1classification
12RELION3D reconstruction
13PHENIX1.19model refinement
CTF correctionType: NONE
Particle selectionNum. of particles selected: 877163
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.68 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 129204 / Symmetry type: POINT
Atomic model buildingB value: 80 / Protocol: FLEXIBLE FIT / Space: REAL / Target criteria: Correlation coefficient / Details: Realspace refinement

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