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- EMDB-3729: RNA polymerase I pre-initiation complex (Pol-Rrn3 focused refinement) -

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Basic information

Entry
Database: EMDB / ID: 3729
TitleRNA polymerase I pre-initiation complex (Pol-Rrn3 focused refinement)
Map data
SampleRNA polymerase I pre-initiation complex with Core Factor, Rrn3 and transcription scaffold
  • RPA1_YEAST DNA-directed RNA polymerase I subunit RPA190
  • RPA2_YEAST DNA-directed RNA polymerase I subunit RPA135
  • RPA49_YEAST DNA-directed RNA polymerase I subunit RPA49
  • RPA43_YEAST DNA-directed RNA polymerase I subunit RPA43
  • RPAC1_YEAST DNA-directed RNA polymerases I and III subunit RPAC1
  • RPA34_YEAST DNA-directed RNA polymerase I subunit RPA34
  • RPAB1_YEAST DNA-directed RNA polymerases I, II, and III subunit RPABC1
  • RPAB2_YEAST DNA-directed RNA polymerases I, II, and III subunit RPABC2
  • RPAC2_YEAST DNA-directed RNA polymerases I and III subunit RPAC2
  • RPAB3_YEAST DNA-directed RNA polymerases I, II, and III subunit RPABC3
  • RPA14_YEAST DNA-directed RNA polymerase I subunit RPA14
  • RPA12_YEAST DNA-directed RNA polymerase I subunit RPA12
  • RPAB4_YEAST DNA-directed RNA polymerases I, II, and III subunit RPABC4
  • RPAB5_YEAST DNA-directed RNA polymerases I, II, and III subunit RPABC5
  • RRN3_YEAST RNA polymerase I-specific transcription initiation factor RRN3
  • (nucleic-acidNucleic acid) x 2
SourceSaccharomyces cerevisiae (baker's yeast)
Methodsingle particle reconstruction / cryo EM / 3.85 Å resolution
AuthorsSadian Y / Tafur L / Kosinski J / Jakobi AJ / Wetzel R / Buczak K / Hagen WJH / Beck M / Sachse C / Muller CW
CitationJournal: EMBO J. / Year: 2017
Title: Structural insights into transcription initiation by yeast RNA polymerase I.
Authors: Yashar Sadian / Lucas Tafur / Jan Kosinski / Arjen J Jakobi / Rene Wetzel / Katarzyna Buczak / Wim Jh Hagen / Martin Beck / Carsten Sachse / Christoph W Müller
Abstract: In eukaryotic cells, RNA polymerase I (Pol I) synthesizes precursor ribosomal RNA (pre-rRNA) that is subsequently processed into mature rRNA. To initiate transcription, Pol I requires the assembly of ...In eukaryotic cells, RNA polymerase I (Pol I) synthesizes precursor ribosomal RNA (pre-rRNA) that is subsequently processed into mature rRNA. To initiate transcription, Pol I requires the assembly of a multi-subunit pre-initiation complex (PIC) at the ribosomal RNA promoter. In yeast, the minimal PIC includes Pol I, the transcription factor Rrn3, and Core Factor (CF) composed of subunits Rrn6, Rrn7, and Rrn11. Here, we present the cryo-EM structure of the 18-subunit yeast Pol I PIC bound to a transcription scaffold. The cryo-EM map reveals an unexpected arrangement of the DNA and CF subunits relative to Pol I. The upstream DNA is positioned differently than in any previous structures of the Pol II PIC. Furthermore, the TFIIB-related subunit Rrn7 also occupies a different location compared to the Pol II PIC although it uses similar interfaces as TFIIB to contact DNA. Our results show that although general features of eukaryotic transcription initiation are conserved, Pol I and Pol II use them differently in their respective transcription initiation complexes.
DateDeposition: May 17, 2017 / Header (metadata) release: May 31, 2017 / Map release: Jul 26, 2017 / Last update: Sep 27, 2017

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.006
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.006
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: : PDB-5oa1
  • Surface level: 0.006
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

Fileemd_3729.map.gz (map file in CCP4 format, 95552 KB)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
288 pix
1.35 Å/pix.
= 388.8 Å
288 pix
1.35 Å/pix.
= 388.8 Å
288 pix
1.35 Å/pix.
= 388.8 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.35 Å
Density
Contour Level:0.006 (by author), 0.006 (movie #1):
Minimum - Maximum-0.19132352 - 0.27216372
Average (Standard dev.)0.0001855439 (0.0046788706)
Details

EMDB XML:

Space Group Number1
Map Geometry
Axis orderXYZ
Dimensions288288288
Origin000
Limit287287287
Spacing288288288
CellA=B=C: 388.80002 Å
α=β=γ: 90 deg.

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.351.351.35
M x/y/z288288288
origin x/y/z0.0000.0000.000
length x/y/z388.800388.800388.800
α/β/γ90.00090.00090.000
start NX/NY/NZ
NX/NY/NZ
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS288288288
D min/max/mean-0.1910.2720.000

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Supplemental data

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Sample components

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Entire RNA polymerase I pre-initiation complex with Core Factor, Rrn3 an...

EntireName: RNA polymerase I pre-initiation complex with Core Factor, Rrn3 and transcription scaffold
Number of components: 18

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Component #1: protein, RNA polymerase I pre-initiation complex with Core Factor...

ProteinName: RNA polymerase I pre-initiation complex with Core Factor, Rrn3 and transcription scaffold
Recombinant expression: No
SourceSpecies: Saccharomyces cerevisiae (baker's yeast)

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Component #2: protein, RPA1_YEAST DNA-directed RNA polymerase I subunit RPA190

ProteinName: RPA1_YEAST DNA-directed RNA polymerase I subunit RPA190
Recombinant expression: No

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Component #3: protein, RPA2_YEAST DNA-directed RNA polymerase I subunit RPA135

ProteinName: RPA2_YEAST DNA-directed RNA polymerase I subunit RPA135
Recombinant expression: No

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Component #4: protein, RPA49_YEAST DNA-directed RNA polymerase I subunit RPA49

ProteinName: RPA49_YEAST DNA-directed RNA polymerase I subunit RPA49
Recombinant expression: No

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Component #5: protein, RPA43_YEAST DNA-directed RNA polymerase I subunit RPA43

ProteinName: RPA43_YEAST DNA-directed RNA polymerase I subunit RPA43
Recombinant expression: No

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Component #6: protein, RPAC1_YEAST DNA-directed RNA polymerases I and III subun...

ProteinName: RPAC1_YEAST DNA-directed RNA polymerases I and III subunit RPAC1
Recombinant expression: No

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Component #7: protein, RPA34_YEAST DNA-directed RNA polymerase I subunit RPA34

ProteinName: RPA34_YEAST DNA-directed RNA polymerase I subunit RPA34
Recombinant expression: No

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Component #8: protein, RPAB1_YEAST DNA-directed RNA polymerases I, II, and III ...

ProteinName: RPAB1_YEAST DNA-directed RNA polymerases I, II, and III subunit RPABC1
Recombinant expression: No

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Component #9: protein, RPAB2_YEAST DNA-directed RNA polymerases I, II, and III ...

ProteinName: RPAB2_YEAST DNA-directed RNA polymerases I, II, and III subunit RPABC2
Recombinant expression: No

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Component #10: protein, RPAC2_YEAST DNA-directed RNA polymerases I and III subun...

ProteinName: RPAC2_YEAST DNA-directed RNA polymerases I and III subunit RPAC2
Recombinant expression: No

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Component #11: protein, RPAB3_YEAST DNA-directed RNA polymerases I, II, and III ...

ProteinName: RPAB3_YEAST DNA-directed RNA polymerases I, II, and III subunit RPABC3
Recombinant expression: No

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Component #12: protein, RPA14_YEAST DNA-directed RNA polymerase I subunit RPA14

ProteinName: RPA14_YEAST DNA-directed RNA polymerase I subunit RPA14
Recombinant expression: No

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Component #13: protein, RPA12_YEAST DNA-directed RNA polymerase I subunit RPA12

ProteinName: RPA12_YEAST DNA-directed RNA polymerase I subunit RPA12
Recombinant expression: No

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Component #14: protein, RPAB4_YEAST DNA-directed RNA polymerases I, II, and III ...

ProteinName: RPAB4_YEAST DNA-directed RNA polymerases I, II, and III subunit RPABC4
Recombinant expression: No

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Component #15: protein, RPAB5_YEAST DNA-directed RNA polymerases I, II, and III ...

ProteinName: RPAB5_YEAST DNA-directed RNA polymerases I, II, and III subunit RPABC5
Recombinant expression: No

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Component #16: protein, RRN3_YEAST RNA polymerase I-specific transcription initi...

ProteinName: RRN3_YEAST RNA polymerase I-specific transcription initiation factor RRN3
Recombinant expression: No

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Component #17: nucleic-acid, non-template DNA strand

Nucleic-acidName: non-template DNA strand / Class: DNA / Structure: OTHER / Synthetic: No
Sequence:
GGTTTAGTCA TGGAGTACAA GTGTGAGGAA AAGTAGTTGG CGTAGCAGGA GAAGTAAAGC AGTTGAAGAC

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Component #18: nucleic-acid, template DNA strand

Nucleic-acidName: template DNA strand / Class: DNA / Structure: OTHER / Synthetic: No
Sequence:
CCAAATCAGT ACCTCATGTT CACACTCCTT TTCATCAACC CTCCATGAAG TACGCTTTCG TCAACTTCTG

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Experimental details

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Sample preparation

SpecimenSpecimen state: particle / Method: cryo EM
Sample solutionSpecimen conc.: 0.2 mg/ml / pH: 7.5
VitrificationInstrument: FEI VITROBOT MARK II / Cryogen name: ETHANE / Humidity: 100 %

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
ImagingMicroscope: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 2 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 105000 X (nominal) / Imaging mode: BRIGHT FIELD / Defocus: 750 - 4000 nm
Specimen HolderModel: OTHER
CameraDetector: GATAN K2 (4k x 4k)

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Image acquisition

Image acquisitionNumber of digital images: 4235

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Image processing

ProcessingMethod: single particle reconstruction / Number of projections: 38589
3D reconstructionSoftware: RELION / Resolution: 3.85 Å / Resolution method: FSC 0.143 CUT-OFF

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