EMDB-11741: Cryo-EM structure of human RNA Polymerase III, map G

Basic information

• Entry: Database: EMDB / ID: EMD-11741
• Title: Cryo-EM structure of human RNA Polymerase III, map G
• Map data: Cryo-EM map of human RNA Polymerase III, map G, unsharpened

Sample

• Complex: Human RNA Polymerase III

Function / homology

Function:

snRNA transcription by RNA polymerase III / RNA Polymerase III Chain Elongation / RNA Polymerase III Transcription Termination / calcitonin gene-related peptide receptor activity / DNA/RNA hybrid binding / regulation of transcription by RNA polymerase I / regulation of transcription by RNA polymerase III / RPAP3/R2TP/prefoldin-like complex / DNA polymerase III complex / Cytosolic sensors of pathogen-associated DNA / RNA Polymerase III Transcription Initiation From Type 1 Promoter / RNA Polymerase III Transcription Initiation From Type 2 Promoter / RNA Polymerase III Transcription Initiation From Type 3 Promoter / RNA Polymerase III Abortive And Retractive Initiation / positive regulation of innate immune response / Abortive elongation of HIV-1 transcript in the absence of Tat / nucleobase-containing compound metabolic process / RNA Polymerase I Transcription Termination / FGFR2 alternative splicing / MicroRNA (miRNA) biogenesis / Signaling by FGFR2 IIIa TM / Viral Messenger RNA Synthesis / RNA Pol II CTD phosphorylation and interaction with CE during HIV infection / RNA Pol II CTD phosphorylation and interaction with CE / Formation of the Early Elongation Complex / Formation of the HIV-1 Early Elongation Complex / mRNA Capping / PIWI-interacting RNA (piRNA) biogenesis / termination of RNA polymerase III transcription / HIV Transcription Initiation / RNA Polymerase II HIV Promoter Escape / Transcription of the HIV genome / 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 / RNA polymerase III activity / transcription initiation at RNA polymerase III promoter / mRNA Splicing - Minor Pathway / RNA Polymerase I Transcription Initiation / Pausing and recovery of Tat-mediated HIV elongation / Tat-mediated HIV elongation arrest and recovery / transcription by RNA polymerase I / HIV elongation arrest and recovery / Pausing and recovery of HIV elongation / Processing of Capped Intron-Containing Pre-mRNA / transcription by RNA polymerase III / RNA polymerase II transcribes snRNA genes / neuropeptide signaling pathway / Tat-mediated elongation of the HIV-1 transcript / transcription elongation by RNA polymerase I / Formation of HIV-1 elongation complex containing HIV-1 Tat / tRNA transcription by RNA polymerase III / RNA polymerase I activity / RNA polymerase I complex / RNA polymerase III complex / Formation of HIV elongation complex in the absence of HIV Tat / RNA polymerase II, core complex / RNA Polymerase II Transcription Elongation / Formation of RNA Pol II elongation complex / RNA Polymerase II Pre-transcription Events / Inhibition of DNA recombination at telomere / positive regulation of interferon-beta production / mRNA Splicing - Major Pathway / acrosomal vesicle / protein-DNA complex / TP53 Regulates Transcription of DNA Repair Genes / RNA Polymerase I Promoter Escape / Transcriptional regulation by small RNAs / NoRC negatively regulates rRNA expression / B-WICH complex positively regulates rRNA expression / Transcription-Coupled Nucleotide Excision Repair (TC-NER) / Formation of TC-NER Pre-Incision Complex / ribonucleoside binding / fibrillar center / DNA-directed 5'-3' RNA polymerase activity / Activation of anterior HOX genes in hindbrain development during early embryogenesis / DNA-directed RNA polymerase / Dual incision in TC-NER / Gap-filling DNA repair synthesis and ligation in TC-NER / single-stranded DNA binding / 4 iron, 4 sulfur cluster binding / double-stranded DNA binding / defense response to virus / Estrogen-dependent gene expression / transcription by RNA polymerase II / cell population proliferation / nucleic acid binding / nuclear body / protein dimerization activity / protein stabilization / intracellular membrane-bounded organelle / innate immune response / nucleotide binding / centrosome / DNA-templated transcription / chromatin binding / magnesium ion binding / DNA binding / zinc ion binding

Domain/homology:

DNA-directed RNA polymerase III subunit RPC5, C-terminal / DNA-directed RNA polymerase III subunit RPC5 C-terminal / 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 / RNA polymerase Rpb4/RPC9, core / DNA-directed RNA-polymerase II subunit / Rpb4/RPC9 superfamily / RNA polymerase subunit Rpb4/RPC9 / RNA polymerase Rpb4 / Zinc finger TFIIS-type signature. / 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 4 / RNA polymerase Rpb2, domain 4 / RNA polymerase Rpb2, domain 5 / RNA polymerase Rpb2, domain 5 / DNA-directed RNA polymerase, M/15kDa subunit / RNA polymerases M/15 Kd subunit / RNA polymerase subunit 9 / 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 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, 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. / Zinc finger, TFIIS-type / DNA-directed RNA polymerase subunit Rpo5/Rpb5 / 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 / RNA polymerase subunit RPABC4/transcription elongation factor Spt4 / 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 Rpb1, domain 3 superfamily / RPB6/omega subunit-like superfamily / RNA polymerase Rpb1, clamp domain superfamily / RNA polymerase Rpb1, domain 3 / RNA polymerase Rpb1, domain 3 / DNA-directed RNA polymerase, subunit beta-prime / RNA polymerase Rpb1, domain 1 / RNA polymerase Rpb1, domain 1 / RNA polymerase, alpha subunit / RNA polymerase Rpb1, domain 5

Component:

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 polymerase III subunit RPC9 / DNA-directed RNA polymerase III subunit RPC4 / DNA-directed RNA polymerases I and III subunit RPAC2 / DNA-directed RNA polymerases I, II, and III subunit RPABC1 / DNA-directed RNA polymerases I, II, and III subunit RPABC3 / DNA-directed RNA polymerases I, II, and III subunit RPABC4 / DNA-directed RNA polymerases I, II, and III subunit RPABC2 / DNA-directed RNA polymerases I, II, and III subunit RPABC5 / DNA-directed RNA polymerase III subunit RPC3 / DNA-directed RNA polymerase III subunit RPC6 / DNA-directed RNA polymerase III subunit RPC5 / DNA-directed RNA polymerase III subunit RPC2 / DNA-directed RNA polymerase III subunit RPC10 / DNA-directed RNA polymerase III subunit RPC8

• Biological species: Homo sapiens (human)
• Method: single particle reconstruction / cryo EM / Resolution: 2.9 Å
• Authors: Girbig M / Misiaszek AD / Vorlaender MK / Mueller CW
• Citation: Journal: Nat Struct Mol Biol / Year: 2021; Title: Cryo-EM structures of human RNA polymerase III in its unbound and transcribing states.; Authors: Mathias Girbig / Agata D Misiaszek / Matthias K Vorländer / Aleix Lafita / Helga Grötsch / Florence Baudin / Alex Bateman / Christoph W Müller / ; Abstract: RNA polymerase III (Pol III) synthesizes transfer RNAs and other short, essential RNAs. Human Pol III misregulation is linked to tumor transformation, neurodegenerative and developmental disorders, and increased sensitivity to viral infections. Here, we present cryo-electron microscopy structures at 2.8 to 3.3 Å resolution of transcribing and unbound human Pol III. We observe insertion of the TFIIS-like subunit RPC10 into the polymerase funnel, providing insights into how RPC10 triggers transcription termination. Our structures resolve elements absent from Saccharomyces cerevisiae Pol III such as the winged-helix domains of RPC5 and an iron-sulfur cluster, which tethers the heterotrimer subcomplex to the core. The cancer-associated RPC7α isoform binds the polymerase clamp, potentially interfering with Pol III inhibition by tumor suppressor MAF1, which may explain why overexpressed RPC7α enhances tumor transformation. Finally, the human Pol III structure allows mapping of disease-related mutations and may contribute to the development of inhibitors that selectively target Pol III for therapeutic interventions.

History

Deposition	Sep 17, 2020	-
Header (metadata) release	Feb 3, 2021	-
Map release	Feb 3, 2021	-
Update	Feb 24, 2021	-
Current status	Feb 24, 2021	Processing site: PDBe / Status: Released

Map

• File: Download / File: emd_11741.map.gz / Format: CCP4 / Size: 144.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
• Annotation: Cryo-EM map of human RNA Polymerase III, map G, unsharpened
• Voxel size: X=Y=Z: 1.05 Å

Density

Contour Level	By AUTHOR: 0.2 / Movie #1: 0.2
Minimum - Maximum	-0.44800106 - 1.5732319
Average (Standard dev.)	0.0079286555 (±0.053539388)

• Symmetry: Space group: 1

Details

EMDB XML:

Map geometry	Axis order X Y Z Origin 0 0 0 Dimensions 336 336 336 Spacing 336 336 336
Cell	A=B=C: 352.8 Å α=β=γ: 90.0 °

CCP4 map header:

mode	Image stored as Reals
Å/pix. X/Y/Z	1.05	1.05	1.05
M x/y/z	336	336	336
origin x/y/z	0.000	0.000	0.000
length x/y/z	352.800	352.800	352.800
α/β/γ	90.000	90.000	90.000
start NX/NY/NZ	0	0	0
NX/NY/NZ	300	300	300
MAP C/R/S	1	2	3
start NC/NR/NS	0	0	0
NC/NR/NS	336	336	336
D min/max/mean	-0.448	1.573	0.008

Supplemental data

Mask #1

• File: emd_11741_msk_1.map

Additional map: Cryo-EM map of human RNA Polymerase III, map...

• File: emd_11741_additional_1.map
• Annotation: Cryo-EM map of human RNA Polymerase III, map G, locally sharpened via LocalDeblur

Half map: #2

• File: emd_11741_half_map_1.map

Half map: #1

• File: emd_11741_half_map_2.map

Sample components

Entire : Human RNA Polymerase III

• Entire: Name: Human RNA Polymerase III

Components

• Complex: Human RNA Polymerase III

Supramolecule #1: Human RNA Polymerase III

• Supramolecule: Name: Human RNA Polymerase III / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#20
• Source (natural): Organism: Homo sapiens (human) / Strain: HEK293F
• Molecular weight: Theoretical: 776 kDa/nm

Experimental details

Structure determination

• Method: cryo EM
• Processing: single particle reconstruction
• Aggregation state: particle

Sample preparation

• Concentration: 2 mg/mL

Buffer

pH: 7.5
Component:

Concentration	Name
15.0 mM	HEPES
150.0 mM	ammonium sulfate
5.0 mM	magnesium chloride
10.0 mM	DTT
4.0 mM	CHAPSO

• Vitrification: Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV / Details: Wait time 10 s Blot force 4 Blot time 4 s.

Electron microscopy

• Microscope: FEI TITAN KRIOS
• Image recording: Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Average electron dose: 40.4 e/Ų; Details: Merged data-set from Micrographs recorded on a Gatan K2 Summit detector and a Gatan K3 detector
• Electron beam: Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
• Electron optics: C2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.25 µm / Nominal defocus min: 0.75 µm / Nominal magnification: 130000
• Sample stage: Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
• Experimental equipment: Model: Titan Krios / Image courtesy: FEI Company

Image processing

• Details: Merged data-set from micrographs recorded on a Gatan K2 Summit detector and a Gatan K3 detector.
• Particle selection: Number selected: 672400 ; Details: Particles that yielded final 3D reconstructions of EMDB entries 11673 and 11736 were merged for this 3D reconstruction.
• CTF correction: Software: (Name: Warp (ver. 1.0.6), RELION (ver. 3.1))
• Startup model: Type of model: INSILICO MODEL / Details: Initial model was generated in cryoSPARC
• Final reconstruction: Applied symmetry - Point group: C₁ (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 2.15.0) / Number images used: 277360
• Initial angle assignment: Type: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
• Final angle assignment: Type: MAXIMUM LIKELIHOOD