EMDB-3218: Structure of transcribing mammalian RNA polymerase II (EC1)

Basic information

• Entry: Database: EMDB / ID: EMD-3218
• Title: Structure of transcribing mammalian RNA polymerase II (EC1)
• Map data: Bovine Pol II elongation complex EC1, B-factor sharpened map

Sample

• Sample: bovine Pol II elongation complex
• Protein or peptide: DNA-directed RNA polymerase II
• Protein or peptide: DNA-directed RNA polymerase II subunit GRINL1A
• Other: DNA-RNA synthetic construct

• Keywords: transcription / elongation

Function / homology

Function:

Formation of RNA Pol II elongation complex / Formation of the Early Elongation Complex / Transcriptional regulation by small RNAs / FGFR2 alternative splicing / RNA polymerase II transcribes snRNA genes / mRNA Capping / mRNA Splicing - Minor Pathway / RNA Polymerase I Transcription Initiation / RNA Polymerase I Promoter Escape / RNA Polymerase II Promoter Escape / RNA Polymerase II Transcription Pre-Initiation And Promoter Opening / RNA Polymerase I Transcription Termination / RNA Polymerase II Transcription Initiation / RNA Polymerase II Transcription Initiation And Promoter Clearance / 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 Pol II CTD phosphorylation and interaction with CE / Estrogen-dependent gene expression / RNA Polymerase II Pre-transcription Events / TP53 Regulates Transcription of DNA Repair Genes / RNA Polymerase II Transcription Elongation / Processing of Capped Intron-Containing Pre-mRNA / B-WICH complex positively regulates rRNA expression / mRNA Splicing - Major Pathway / Formation of TC-NER Pre-Incision Complex / Dual incision in TC-NER / Gap-filling DNA repair synthesis and ligation in TC-NER / DNA/RNA hybrid binding / positive regulation of nuclear-transcribed mRNA poly(A) tail shortening / maintenance of transcriptional fidelity during transcription elongation by RNA polymerase II / nuclear-transcribed mRNA catabolic process / positive regulation of translational initiation / transcription by RNA polymerase III / transcription by RNA polymerase I / RNA polymerase I complex / transcription elongation by RNA polymerase I / RNA polymerase III complex / transcription-coupled nucleotide-excision repair / core promoter sequence-specific DNA binding / RNA polymerase II, core complex / tRNA transcription by RNA polymerase III / : / translation initiation factor binding / DNA-directed RNA polymerase activity / transcription initiation at RNA polymerase II promoter / P-body / euchromatin / protein-DNA complex / ribonucleoside binding / : / : / : / : / : / : / DNA-directed RNA polymerase / single-stranded DNA binding / Hydrolases; Acting on ester bonds; Exoribonucleases producing 5'-phosphomonoesters / transcription by RNA polymerase II / nucleic acid binding / chromosome, telomeric region / single-stranded RNA binding / hydrolase activity / protein dimerization activity / RNA-directed RNA polymerase / RNA-directed RNA polymerase activity / nucleotide binding / chromatin binding / nucleolus / magnesium ion binding / mitochondrion / DNA binding / zinc ion binding / nucleus / cytosol

Domain/homology:

DNA-directed RNA polymerase II subunit Rpb4-like / RNA polymerase Rpb1 C-terminal repeat / RNA polymerase II, heptapeptide repeat, eukaryotic / Eukaryotic RNA polymerase II heptapeptide repeat. / RNA polymerase Rpb1, domain 6 / RNA polymerase Rpb1, domain 6 / RNA polymerase Rpb4/RPC9, core / DNA-directed RNA-polymerase II subunit / RNA polymerase Rpb1, domain 7 / RNA polymerase Rpb1, domain 7 superfamily / RNA polymerase Rpb1, domain 7 / Pol II subunit B9, C-terminal zinc ribbon / RNA polymerase RBP11 / 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 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. / Zinc finger, TFIIS-type / Transcription factor S-II (TFIIS) / Zinc finger TFIIS-type profile. / C2C2 Zinc finger / RNA polymerase subunit RPABC4/transcription elongation factor Spt4 / 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. / Ribosomal protein S1-like RNA-binding domain / S1 RNA binding domain / S1 domain / DNA-directed RNA polymerase, subunit beta-prime / 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 / RNA polymerase, beta subunit, protrusion / RNA polymerase beta subunit / RNA polymerase, alpha subunit / RNA polymerase Rpb1, domain 5 / RNA polymerase Rpb1, domain 4 / RNA polymerase Rpb1, domain 2 / RNA polymerase Rpb1, domain 5 / RNA polymerase Rpb1, domain 4 / DNA-directed RNA polymerase, insert domain superfamily

Component:

DNA-directed RNA polymerase II subunit RPB2 / DNA-directed RNA polymerases I, II, and III subunit RPABC3 / DNA-directed RNA polymerase II subunit RPB1 / DNA-directed RNA polymerase II subunit RPB4 / DNA-directed RNA polymerases I, II, and III subunit RPABC1 / DNA-directed RNA polymerase II subunit RPB9 / DNA-directed RNA polymerases I, II, and III subunit RPABC5 / DNA-directed RNA polymerase II subunit RPB11 / DNA-directed RNA polymerases I, II, and III subunit RPABC2 / DNA-directed RNA polymerase II subunit RPB3 / DNA-directed RNA polymerases I, II, and III subunit RPABC4 / DNA-directed RNA polymerase II subunit RPB7

• Biological species: Bos taurus (domestic cattle) / Homo sapiens (human) / unidentified (others)
• Method: single particle reconstruction / cryo EM / Resolution: 3.4 Å
• Authors: Bernecky C / Herzog F / Baumeister W / Plitzko JM / Cramer P
• Citation: Journal: Nature / Year: 2016; Title: Structure of transcribing mammalian RNA polymerase II.; Authors: Carrie Bernecky / Franz Herzog / Wolfgang Baumeister / Jürgen M Plitzko / Patrick Cramer / ; Abstract: RNA polymerase (Pol) II produces messenger RNA during transcription of protein-coding genes in all eukaryotic cells. The Pol II structure is known at high resolution from X-ray crystallography for two yeast species. Structural studies of mammalian Pol II, however, remain limited to low-resolution electron microscopy analysis of human Pol II and its complexes with various proteins. Here we report the 3.4 Å resolution cryo-electron microscopy structure of mammalian Pol II in the form of a transcribing complex comprising DNA template and RNA transcript. We use bovine Pol II, which is identical to the human enzyme except for seven amino-acid residues. The obtained atomic model closely resembles its yeast counterpart, but also reveals unknown features. Binding of nucleic acids to the polymerase involves 'induced fit' of the mobile Pol II clamp and active centre region. DNA downstream of the transcription bubble contacts a conserved 'TPSA motif' in the jaw domain of the Pol II subunit RPB5, an interaction that is apparently already established during transcription initiation. Upstream DNA emanates from the active centre cleft at an angle of approximately 105° with respect to downstream DNA. This position of upstream DNA allows for binding of the general transcription elongation factor DSIF (SPT4-SPT5) that we localize over the active centre cleft in a conserved position on the clamp domain of Pol II. Our results define the structure of mammalian Pol II in its functional state, indicate that previous crystallographic analysis of yeast Pol II is relevant for understanding gene transcription in all eukaryotes, and provide a starting point for a mechanistic analysis of human transcription.

History

Deposition	Oct 26, 2015	-
Header (metadata) release	Nov 11, 2015	-
Map release	Jan 20, 2016	-
Update	Feb 3, 2016	-
Current status	Feb 3, 2016	Processing site: PDBe / Status: Released

Map

• File: Download / File: emd_3218.map.gz / Format: CCP4 / Size: 31.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
• Annotation: Bovine Pol II elongation complex EC1, B-factor sharpened map
• Voxel size: X=Y=Z: 1.35 Å

Density

Contour Level	By AUTHOR: 0.07 / Movie #1: 0.05
Minimum - Maximum	-0.30116436 - 0.4999862
Average (Standard dev.)	0.00011944 (±0.0182527)

• Symmetry: Space group: 1

Details

EMDB XML:

Map geometry	Axis order X Y Z Origin 0 0 0 Dimensions 204 204 204 Spacing 204 204 204
Cell	A=B=C: 275.4 Å α=β=γ: 90.0 °

CCP4 map header:

mode	Image stored as Reals
Å/pix. X/Y/Z	1.35	1.35	1.35
M x/y/z	204	204	204
origin x/y/z	0.000	0.000	0.000
length x/y/z	275.400	275.400	275.400
α/β/γ	90.000	90.000	90.000
MAP C/R/S	1	2	3
start NC/NR/NS	0	0	0
NC/NR/NS	204	204	204
D min/max/mean	-0.301	0.500	0.000

Supplemental data

Supplemental map: emd 3218 additional 1.map

• File: emd_3218_additional_1.map

Sample components

Entire : bovine Pol II elongation complex

• Entire: Name: bovine Pol II elongation complex

Components

• Sample: bovine Pol II elongation complex
• Protein or peptide: DNA-directed RNA polymerase II
• Protein or peptide: DNA-directed RNA polymerase II subunit GRINL1A
• Other: DNA-RNA synthetic construct

Supramolecule #1000: bovine Pol II elongation complex

• Supramolecule: Name: bovine Pol II elongation complex / type: sample / ID: 1000 ; Details: Recombinant human Gdown1 was present during sample preparation but density was not observed in this map.; Number unique components: 3
• Molecular weight: Theoretical: 590 KDa

Macromolecule #1: DNA-directed RNA polymerase II

• Macromolecule: Name: DNA-directed RNA polymerase II / type: protein_or_peptide / ID: 1 / Name.synonym: RNA polymerase II / Number of copies: 1 / Oligomeric state: Monomer / Recombinant expression: No
• Source (natural): Organism: Bos taurus (domestic cattle) / synonym: Cow / Tissue: Thymus
• Molecular weight: Theoretical: 520 KDa
• Sequence: GO: DNA-directed RNA polymerase activity

Macromolecule #2: DNA-directed RNA polymerase II subunit GRINL1A

• Macromolecule: Name: DNA-directed RNA polymerase II subunit GRINL1A / type: protein_or_peptide / ID: 2 / Name.synonym: Gdown1 / Number of copies: 1 / Oligomeric state: Monomer / Recombinant expression: Yes
• Source (natural): Organism: Homo sapiens (human) / synonym: Human
• Molecular weight: Theoretical: 40 KDa
• Recombinant expression: Organism: Escherichia coli (E. coli) / Recombinant strain: BL21(DE3)RIL / Recombinant plasmid: pOPINB

Macromolecule #3: DNA-RNA synthetic construct

• Macromolecule: Name: DNA-RNA synthetic construct / type: other / ID: 3 / Name.synonym: DNA-RNA elongation scaffold / Classification: DNA/RNA / Structure: OTHER / Synthetic?: Yes
• Source (natural): Organism: unidentified (others)
• Molecular weight: Theoretical: 30 KDa

Experimental details

Structure determination

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

Sample preparation

• Concentration: 0.3 mg/mL
• Buffer: pH: 7.25 / Details: 150 mM NaCl, 5 mM HEPES, 0.01 mM ZnCl2, 10 mM DTT
• Grid: Details: Quantifoil R 3.5/1 holey carbon grids
• Vitrification: Cryogen name: ETHANE / Chamber humidity: 100 % / Instrument: FEI VITROBOT MARK IV; Method: Four microliters of sample was applied to glow-discharged Quantifoil R 3.5/1 holey carbon grids, which were then blotted for 8.5s and plunge-frozen in liquid ethane.

Electron microscopy

• Microscope: FEI TITAN KRIOS
• Specialist optics: Energy filter - Name: GIF Quantum / Energy filter - Lower energy threshold: 0.0 eV / Energy filter - Upper energy threshold: 20.0 eV
• Date: Dec 1, 2014
• Image recording: Category: CCD / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Number real images: 1172 / Average electron dose: 43 e/Ų; Details: Each movie image was collected over 8 s fractionated into 40 frames (0.2 s each).
• Electron beam: Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
• Electron optics: Calibrated magnification: 37037 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2 mm / Nominal defocus max: 3.1 µm / Nominal defocus min: 0.6 µm / Nominal magnification: 37000
• Sample stage: Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
• Experimental equipment: Model: Titan Krios / Image courtesy: FEI Company

Image processing

• CTF correction: Details: Each particle
• Final reconstruction: Applied symmetry - Point group: C₁ (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 3.4 Å / Resolution method: OTHER / Software - Name: RELION / Number images used: 264134