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- EMDB-3178: Cryo-EM structure of yeast RNA polymerase III elongation complex ... -

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Basic information

Entry
Database: EMDB / ID: EMD-3178
TitleCryo-EM structure of yeast RNA polymerase III elongation complex at 3.9 A
Map dataReconstruction of elongating RNA polymerase III
Sample
  • Sample: S.cerevisiae RNA polymerase III elongation complex at 3.9 A
  • Protein or peptide: RNA polymerase III
  • Other: Transcription Bubble
KeywordsRNA polymerase III / pol III / transcription / RNA polymerase
Function / homology
Function and homology information


RNA Polymerase I Transcription Initiation / Processing of Capped Intron-Containing Pre-mRNA / RNA Polymerase III Transcription Initiation From Type 2 Promoter / RNA Pol II CTD phosphorylation and interaction with CE / Formation of the Early Elongation Complex / mRNA Capping / RNA polymerase II transcribes snRNA genes / TP53 Regulates Transcription of DNA Repair Genes / RNA Polymerase II Promoter Escape / RNA Polymerase II Transcription Pre-Initiation And Promoter Opening ...RNA Polymerase I Transcription Initiation / Processing of Capped Intron-Containing Pre-mRNA / RNA Polymerase III Transcription Initiation From Type 2 Promoter / RNA Pol II CTD phosphorylation and interaction with CE / Formation of the Early Elongation Complex / mRNA Capping / RNA polymerase II transcribes snRNA genes / TP53 Regulates Transcription of DNA Repair Genes / RNA Polymerase II Promoter Escape / RNA Polymerase II Transcription Pre-Initiation And Promoter Opening / RNA Polymerase II Transcription Initiation / RNA Polymerase II Transcription Initiation And Promoter Clearance / termination of RNA polymerase III transcription / RNA Polymerase II Pre-transcription Events / RNA-templated transcription / Formation of TC-NER Pre-Incision Complex / transcription initiation at RNA polymerase III promoter / RNA Polymerase I Promoter Escape / termination of RNA polymerase I transcription / nucleolar large rRNA transcription by RNA polymerase I / Gap-filling DNA repair synthesis and ligation in TC-NER / transcription initiation at RNA polymerase I promoter / Estrogen-dependent gene expression / transcription by RNA polymerase III / Dual incision in TC-NER / RNA polymerase I complex / transcription elongation by RNA polymerase I / RNA polymerase III complex / RNA polymerase II, core complex / tRNA transcription by RNA polymerase III / transcription by RNA polymerase I / nucleotidyltransferase activity / transcription initiation at RNA polymerase II promoter / transcription elongation by RNA polymerase II / ribonucleoside binding / DNA-directed RNA polymerase / DNA-directed RNA polymerase activity / peroxisome / ribosome biogenesis / single-stranded DNA binding / nucleic acid binding / transcription by RNA polymerase II / protein dimerization activity / nucleotide binding / nucleolus / mitochondrion / DNA binding / zinc ion binding / nucleoplasm / metal ion binding / nucleus / cytoplasm / cytosol
Similarity search - Function
DNA-directed RNA polymerase III subunit RPC4 / RNA polymerase III RPC4 / DNA-directed RNA polymerase III, subunit Rpc31 / DNA-directed RNA polymerase III subunit Rpc31 / Pol III subunit C11, C-terminal zinc ribbon / DNA-directed RNA polymerase III subunit Rpc5 / : / DNA-directed RNA polymerase III subunit RPC1, N-terminal / DNA-directed RNA polymerase III subunit RPC1, C-terminal / RPC5 protein ...DNA-directed RNA polymerase III subunit RPC4 / RNA polymerase III RPC4 / DNA-directed RNA polymerase III, subunit Rpc31 / DNA-directed RNA polymerase III subunit Rpc31 / Pol III subunit C11, C-terminal zinc ribbon / DNA-directed RNA polymerase III subunit Rpc5 / : / DNA-directed RNA polymerase III subunit RPC1, N-terminal / DNA-directed RNA polymerase III subunit RPC1, C-terminal / RPC5 protein / RNA polymerase III, subunit Rpc25 / DNA-directed RNA polymerase III subunit RPC9 / RNA polymerase III subunit Rpc25 / RNA polymerase Rpc34 / RNA polymerase III Rpc82, C -terminal / RNA polymerase Rpc34-like / DNA-directed RNA polymerase III subunit RPC3 / : / RNA polymerase Rpc34 subunit / RNA polymerase III subunit RPC82 / DNA-directed RNA polymerase III subunit RPC3, helical hairpin domain / POLR3C, C-terminal winged-helix domain / RNA polymerase III subunit RPC82-related, helix-turn-helix / RNA polymerase III subunit RPC82 helix-turn-helix domain / DNA-directed RNA polymerase, subunit E/RPC8 / DNA-directed RNA polymerases I and III subunit AC19 / DNA-directed RNA polymerases I and III subunit AC40 / Rpb4/RPC9 superfamily / RNA polymerase subunit Rpb4/RPC9 / RNA polymerase Rpb4 / HRDC-like superfamily / RNA polymerase Rpb7-like , N-terminal / RNA polymerase Rpb7-like, N-terminal domain superfamily / RNA polymerase subunit Rpb7-like / SHS2 domain found in N terminus of Rpb7p/Rpc25p/MJ0397 / RNA polymerase Rpb2, domain 5 / RNA polymerase Rpb2, domain 5 / RNA polymerase Rpb2, domain 4 / RNA polymerase Rpb2, domain 4 / DNA-directed RNA polymerase, M/15kDa subunit / RNA polymerases M/15 Kd subunit / RNA polymerase subunit 9 / DNA-directed RNA polymerase M, 15kDa subunit, conserved site / RNA polymerases M / 15 Kd subunits signature. / DNA-directed RNA polymerase subunit/transcription factor S / : / RNA polymerase, Rpb8 / DNA-directed RNA polymerases I, II, and III subunit RPABC4 / RNA polymerase Rpb8 / RNA polymerase subunit 8 / RNA polymerase, Rpb5, N-terminal / RNA polymerase Rpb5, N-terminal domain superfamily / RNA polymerase Rpb5, N-terminal domain / DNA-directed RNA polymerase, subunit RPB6 / DNA-directed RNA polymerase subunit RPABC5/Rpb10 / RNA polymerases, subunit N, zinc binding site / RNA polymerase subunit RPB10 / RNA polymerases N / 8 kDa subunit / RNA polymerases N / 8 Kd subunits signature. / DNA directed RNA polymerase, 7 kDa subunit / RNA polymerase archaeal subunit P/eukaryotic subunit RPABC4 / RNA polymerase, subunit H/Rpb5, conserved site / RNA polymerases H / 23 Kd subunits signature. / RNA polymerase subunit CX / DNA-directed RNA polymerase, 30-40kDa subunit, conserved site / DNA-directed RNA polymerase subunit Rpo3/Rpb3/RPAC1 / RNA polymerases D / 30 to 40 Kd subunits signature. / DNA-directed RNA polymerase Rpb11, 13-16kDa subunit, conserved site / DNA-directed RNA polymerase subunit Rpo11 / RNA polymerases L / 13 to 16 Kd subunits signature. / RNA polymerase subunit RPABC4/transcription elongation factor Spt4 / Zinc finger, TFIIS-type / Transcription factor S-II (TFIIS) / Zinc finger TFIIS-type profile. / C2C2 Zinc finger / DNA-directed RNA polymerase, RBP11-like dimerisation domain / RNA polymerase Rpb3/Rpb11 dimerisation domain / RNA polymerase, subunit H/Rpb5 C-terminal / DNA-directed RNA polymerase subunit Rpo5/Rpb5 / RPB5-like RNA polymerase subunit superfamily / RNA polymerase Rpb5, C-terminal domain / Archaeal Rpo6/eukaryotic RPB6 RNA polymerase subunit / DNA-directed RNA polymerase, 14-18kDa subunit, conserved site / RNA polymerases K / 14 to 18 Kd subunits signature. / RNA polymerase Rpb6 / RNA polymerase, subunit omega/Rpo6/RPB6 / RNA polymerase Rpb6 / RNA polymerase Rpb2, domain 2 superfamily / RNA polymerase Rpb1, domain 3 superfamily / RPB6/omega subunit-like superfamily / DNA-directed RNA polymerase, insert domain / DNA-directed RNA polymerase, RpoA/D/Rpb3-type / RNA polymerase Rpb3/RpoA insert domain / RNA polymerase Rpb3/Rpb11 dimerisation domain / RNA polymerases D / RNA polymerase Rpb1, clamp domain superfamily / RNA polymerase Rpb1, domain 3 / RNA polymerase Rpb1, domain 3 / RNA polymerase Rpb1, domain 1 / RNA polymerase Rpb1, domain 1
Similarity search - Domain/homology
DNA-directed RNA polymerase III subunit RPC1 / DNA-directed RNA polymerases I and III subunit RPAC1 / DNA-directed RNA polymerase III subunit RPC7 / DNA-directed RNA polymerases I, II, and III subunit RPABC1 / DNA-directed RNA polymerases I, II, and III subunit RPABC2 / DNA-directed RNA polymerases I, II, and III subunit RPABC3 / DNA-directed RNA polymerases I, II, and III subunit RPABC5 / DNA-directed RNA polymerase III subunit RPC2 / DNA-directed RNA polymerase III subunit RPC4 / DNA-directed RNA polymerases I and III subunit RPAC2 ...DNA-directed RNA polymerase III subunit RPC1 / DNA-directed RNA polymerases I and III subunit RPAC1 / DNA-directed RNA polymerase III subunit RPC7 / DNA-directed RNA polymerases I, II, and III subunit RPABC1 / DNA-directed RNA polymerases I, II, and III subunit RPABC2 / DNA-directed RNA polymerases I, II, and III subunit RPABC3 / DNA-directed RNA polymerases I, II, and III subunit RPABC5 / DNA-directed RNA polymerase III subunit RPC2 / DNA-directed RNA polymerase III subunit RPC4 / DNA-directed RNA polymerases I and III subunit RPAC2 / DNA-directed RNA polymerase III subunit RPC3 / DNA-directed RNA polymerase III subunit RPC6 / DNA-directed RNA polymerase III subunit RPC8 / DNA-directed RNA polymerase III subunit RPC5 / DNA-directed RNA polymerases I, II, and III subunit RPABC4 / DNA-directed RNA polymerase III subunit RPC9 / DNA-directed RNA polymerase III subunit RPC10
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.9 Å
AuthorsHoffmann NA / Jakobi AJ / Moreno-Morcillo M / Glatt S / Kosinski J / Hagen WJ / Sachse C / Muller CW
CitationJournal: Nature / Year: 2015
Title: Molecular structures of unbound and transcribing RNA polymerase III.
Authors: Niklas A Hoffmann / Arjen J Jakobi / María Moreno-Morcillo / Sebastian Glatt / Jan Kosinski / Wim J H Hagen / Carsten Sachse / Christoph W Müller /
Abstract: Transcription of genes encoding small structured RNAs such as transfer RNAs, spliceosomal U6 small nuclear RNA and ribosomal 5S RNA is carried out by RNA polymerase III (Pol III), the largest yet ...Transcription of genes encoding small structured RNAs such as transfer RNAs, spliceosomal U6 small nuclear RNA and ribosomal 5S RNA is carried out by RNA polymerase III (Pol III), the largest yet structurally least characterized eukaryotic RNA polymerase. Here we present the cryo-electron microscopy structures of the Saccharomyces cerevisiae Pol III elongating complex at 3.9 Å resolution and the apo Pol III enzyme in two different conformations at 4.6 and 4.7 Å resolution, respectively, which allow the building of a 17-subunit atomic model of Pol III. The reconstructions reveal the precise orientation of the C82-C34-C31 heterotrimer in close proximity to the stalk. The C53-C37 heterodimer positions residues involved in transcription termination close to the non-template DNA strand. In the apo Pol III structures, the stalk adopts different orientations coupled with closed and open conformations of the clamp. Our results provide novel insights into Pol III-specific transcription and the adaptation of Pol III towards its small transcriptional targets.
History
DepositionOct 2, 2015-
Header (metadata) releaseNov 4, 2015-
Map releaseNov 25, 2015-
UpdateJan 13, 2016-
Current statusJan 13, 2016Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.06
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.06
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-5fj8
  • Surface level: 0.06
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_3178.jpg

Downloads & links

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Map

FileDownload / File: emd_3178.map.gz / Format: CCP4 / Size: 96.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of elongating RNA polymerase III
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.08 Å/pix.
x 296 pix.
= 320.864 Å		1.08 Å/pix.
x 296 pix.
= 320.864 Å		1.08 Å/pix.
x 296 pix.
= 320.864 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.084 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.06 / Movie #1: 0.06
Minimum - Maximum-0.32748002 - 0.53550452
Average (Standard dev.)0.00124018 (±0.01496994)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions296296296
Spacing296296296
CellA=B=C: 320.86398 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0841.0841.084
M x/y/z296296296
origin x/y/z0.0000.0000.000
length x/y/z320.864320.864320.864
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS296296296
D min/max/mean-0.3270.5360.001

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Supplemental data

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Sample components

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Entire : S.cerevisiae RNA polymerase III elongation complex at 3.9 A

EntireName: S.cerevisiae RNA polymerase III elongation complex at 3.9 A
Components
  • Sample: S.cerevisiae RNA polymerase III elongation complex at 3.9 A
  • Protein or peptide: RNA polymerase III
  • Other: Transcription Bubble

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Supramolecule #1000: S.cerevisiae RNA polymerase III elongation complex at 3.9 A

SupramoleculeName: S.cerevisiae RNA polymerase III elongation complex at 3.9 A
type: sample / ID: 1000 / Oligomeric state: Monomer / Number unique components: 20
Molecular weightTheoretical: 720 KDa

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Macromolecule #1: RNA polymerase III

MacromoleculeName: RNA polymerase III / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Oligomeric state: Monomer / Recombinant expression: No
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: Yeast
Molecular weightExperimental: 690 KDa

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Macromolecule #2: Transcription Bubble

MacromoleculeName: Transcription Bubble / type: other / ID: 2 / Name.synonym: template DNA, non-template DNA, RNA / Classification: DNA/RNA / Structure: OTHER / Synthetic?: Yes
Source (natural)Organism: synthetic construct (others)
Molecular weightExperimental: 290 KDa

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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

Concentration0.2 mg/mL
BufferpH: 7.5 / Details: 150mM (NH4)2SO4, 15mM Tris, 10mM DTT, 5mM MgCl2
GridDetails: Cu 400 mesh Quantifoil 1.2/1.3
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Instrument: FEI VITROBOT MARK III
Method: 2.5 ul of sample was applied 15 seconds wait time Blot for 9 seconds before plunging

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Electron microscopy

MicroscopeFEI TITAN KRIOS
DateOct 15, 2014
Image recordingCategory: CCD / Film or detector model: FEI FALCON II (4k x 4k) / Number real images: 2639 / Average electron dose: 45 e/Å2
Details: Seven frames were recorded with a dose of 6.5 e/A2 per frame.
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 4.2 µm / Nominal defocus min: 1.4 µm / Nominal magnification: 75000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: Each particle
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.9 Å / Resolution method: OTHER / Software - Name: Relion / Number images used: 49543
FSC plot (resolution estimation)

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