2.2.1 Expression and purification of human Gdown1 (hGdown1)
Purification of hGdown1 was performed as described before (Bernecky et al., 2016a). Gene-optimized hGdown1 (Life Technologies) was cloned into pOPINB (N-terminal His6 tag and 3C protease site). The vector was transformed to BL21(DE3)RIL competent cells and plated on LB agar plate and cultured overnight in 37°C incubator. Single colony was picked and
cultured in LB medium with kanamycin and chloramphenicol overnight at 37°C while shaking at 160rpm. The overnight E.coli cells were cultured in 2L LB medium (with kanamycin and chloramphenicol) at 37°C for 3 to 4 hours till the OD600 arriving to 0.6 to 0.8, then the protein was expressed by inducing with 0.5mM IPTG for 3 to 4 hours at 37°C. The cells were harvested at a speed of 6000rpm for 15 minutes. The supernatant was gently discarded and
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the pellet was re-suspended in hGdown1 lysis buffer, frozen in liquid nitrogen, and kept at 80°C for purification.
For purification, the re-suspended cells were transferred to a metal beaker for sonication with power 20%, 0.6 on, 0.4 off settings for 10 minutes. The sonicated material was
transferred to 2 centrifugation tubes and spun down for 30 minutes at 4° with Beckman A27 rotor and a speed of 15,000rpm. The supernatant was transferred to a new tube and filtered with 0.8uM filter. The filtered supernatant was loaded to 5ml HisTrap™ High Performance column (GE Healthcare) which was pre-equilibrated with hGdown1 lysis buffer. The column was washed with 10 CV ofhGdown1 lysis buffer, 5CV of hGdown1 wash buffer 1 and eluted with 5CV of hGdown1 elution buffer. The eluted protein was mixed with TEV protease (1:10 ratio of TEV and protein) and dialyzed to hGdown1 lysis buffer overnight. The next day the protein was centrifuged at 27,000rpm for 10 minutes to remove the possible precipitation after cleavage. The supernatant was loaded to Ni column which was equilibrated with lysis buffer beforehand. The flow through was collected, the column was washed with hGdown1 wash buffer 2 and the washed buffer was also collected. The protein was eluted from Ni column and loaded to monoS (GE Healthcare) column. Column was washed for 10CV with wash buffer 1 and eluted with a NaCl gradient from 0M to 1M. The peak fractions were identified with SDS-PAGE. The target protein was pooled, concentrated and loaded to gel filtration. Column Superdex 200 10/300 GL (GE Healthcare) was used for gel filtration. The peak fractions were identified again with SDS-PAGE and the target fractions were pooled and concentrated to 2 to 3mg/ml with Amicon Ultra-15 Centrifugal Filter Unit (10 kDa MWCO) (Merck KGaA, Germany). The final protein solution was centrifuged at maximum speed for 2 minutes and aliquoted as 5ul aliquots, frozen in liquid nitrogen and stored at -80°C ready for use.
2.2.2 Purification of Sus scrofa Pol II
Sus scrofa Pol II was purified essentially as described for the bovine Pol II preparation (Hodo and Blatti, 1977; Thompson et al., 1990). 500g frozen pig thymus were crashed into pieces with a hammer. The broken pieces were added to a pre-chilled Warning blender with 1L 0M HepR buffer and homogenized on high speed for 3 minutes. The homogenized material was centrifuged with SLA-1500 rotor at 11,000rpm for 20 minutes at 4°C. Unless special
emphasis, all the steps below were carried out at 4°C. The supernatant was filtered with 2 layer of miracloth into a chilled glass graduated cylinder and then transferred to a chilled 2L beaker with stirring bar. 5% polyethylenimine (PEI,Sigma-Aldrich) was slowly added to a final concentration of 0.02% while stirring. The stirring was kept at 4°C for at least 10 minutes. Then the precipitated material was transferred to the centrifugation tubes and centrifuged with SLA-1500 rotor at 11,000rpm for 20 minutes. The pellet from the centrifugation was fully re-suspended with 0.15M HepR buffer and centrifuged with SLA-1500 rotor at 11,000rpm for 20 minutes. At the same time, the MacroPrep Q column was washed with 2 column volume (CV) water, 2CV 0M HepR buffer, 3CV 0.6M HepR buffer and equilibrated with 2CV 0.2M HepR buffer. After centrifugation, the supernatant was adjusted to the conductivity of 0.2M HepR buffer and loaded to MacroPrep Q column with a very slow
15
flow rate (gravity flow). After loading, the column was washed with 3CV of 0.2M HepR buffer before eluting with 3CV of 0.4M HepR buffer. The eluted fraction was precipitated slowly with finely ground ammonium sulfate till saturation while stirring. The stirring was kept for at least one hour at 4°C before centrifuging with F14 rotor at 15,000rpm for 30 minutes.
After centrifugation, the supernatant was gently removed and the pellet was dissolved in 0M HepR buffer with 1mM DTT. This was named as ‘Ab input’. The conductivity of ‘Ab input’ was adjusted to match the conductivity of 0.15M HepR buffer and followed by a centrifugation at 15,000rpm for 30 minutes. The 8WG16 (αRPB1 CTD) antibody-coupled Sepharose column was equilibrated with 0.15M HepR buffer and the Ab input was loaded to the antibody column in gravity flow (the beads bed was not allowed to be disturbed during the whole process). The column was washed with 0.5M HepR buffer (also in gravity flow) and then moved to room temperature. The antibody column was kept at room temperature for at least 15 minutes to make sure the resin is at room temperature. Then the protein was eluted with 0.5M HepR buffer plus 50% (v/v) glycerol. The eluted drops were collected with 50ml conical tubes containing 20ml dilution buffer. The elution was fractionated every 5ml, in total 5 fractions were collected. After elution, all the fractions were identified with SDS-PAGE. Fractions with Pol II were collected and loaded to UnoQ column (Biorad) which was equilibrated with 0.1M HepR buffer beforehand. UnoQ column was washed with 5CV of 0.1M HepR buffer and eluted with a linear gradient from 0.1M HepR to 0.5M HepR. The peak fractions were taken and loaded to SDS-PAGE gel. Fractions without pig Gdown1 were pooled, 3-fold molar excess of hGdown1 was added and kept on ice for 2 to 3 hours. Then the sample was loaded to a HiPrep 16/60 Sephacryl S-300 HR column (GE Healthcare, Little Chalfont, United Kingdom). Peak fractions were identified with SDS-PAGE and fractions containing the Pol II-hGdown1 complex were collected and concentrated to a concentration of 2-3 mg/ml using an Amicon Ultra-15 Centrifugal Filter Unit (100 kDa MWCO) (Merck KGaA, Germany). Sample aliquots were snap frozen in liquid nitrogen and stored at -80 °C prior to use. The typical yield is about 2-4 mg from ~500 g pig thymus.
2.2.3 SDS-PAGE
SDS-PAGE was performed by using pre-cast NuPAGE Bis-Tris 4-12% gels (Invitrogen). 4x NuPAGE LDS loading buffer (Invitrogen) was added to the Protein sample to a final concentration of 1x. The samples were boiled at 95°C for 5 to 10 minutes and loaded carefully to the wells of the gel. At least one well of one gel should be loaded with protein marker (precision plus proteinTM Dual Color Standards, BIO-RAD). Gels were run in either 1xMES or 1xMOPS buffer (diluted from NuPAGE 20 x stock, Invitrogen. For small proteins, MOPS buffer has better resolution) for 30 to 60 minutes at 200V. After running, gels were taken out and stained with InstantBlue (Expedeon). The destaining of the gels was
performed with water and the gel was scanned with Epson Perfection V700 Photo Fachbettscanner.
2.2.4 Formation of elongation complex (EC)
The DNA scaffold used for the EC is the same as the one used for the bovine RNA
polymerase II-DSIF complex (Bernecky et al., 2016a). A 20nt RNA was used for the formation
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of a 9nt DNA-RNA hybrid and 11nt of exiting RNA. The template DNA/RNA was annealed (Brueckner et al., 2007) and a 1.5 fold molar excess of scaffold was added to the Pol
II-hGdown1 complex. The sample was incubated on ice for 10 min and subsequently incubated for an additional 15 min at 20 °C while shaking at 550rpm. Then the non-template DNA was added and the sample was kept at 20°C for another 20 minutes. The complex was
crosslinked with 3mM BS3 (Thermo Scientific, final concentration) on ice for 30 min. The crosslinking reaction was quenched with 50mM ammonium bicarbonate and applied to a Superdex 200 increase 10/300 GL column (GE Healthcare) pre-equilibrated with S-300 buffer. The peak containing the complex was pooled and concentrated to a concentration of 473 µM. A 1.5-fold molar excess of α-amanitin was added to the elongation complex. The sample was incubated on ice for 20 min and then loaded directly to the grids.
2.2.5 Electron microscopy
4 µL of the protein complex solution was applied to glow-discharged Quantifoil R2/2 gold grids (Quantifoil) and plunged into liquid ethane after blotting with a FEI Vitrobot Mark IV (FEI, Hillsboro, USA). Images were acquired on a FEI Titan Krios, operated at 300 keV and equipped with a Gatan K2 Summit direct electron detector and a Quantum GIF. Micrographs were collected automatically with the software package EPU (FEI) at a nominal magnification of 130k (1.07 Å per pixel) in counting mode. The dose rate was 3.8 e-/pixel/s. Three images were acquired per foil hole. Each micrograph was collected with a total dose of 35 electrons per square angstrom over a 10-second exposure, fractionated into 40 frames (0.25 s each).
Defocus values ranged from −1 to −3 µm. Micrograph frames were aligned and corrected with MmotionCcorr2 (Zheng et al., 2017). Unless otherwise noted, data processing was performed using RELION 2.1 (Fernandez-Leiro and Scheres, 2017). Contrast transfer function (CTF) parameters were estimated using Gctf (Zhang, 2016). Initial 2D classes were calculated from 2,909 manually selected particles from 37 micrographs. The initial 2D classes were used as templates for auto-picking. After manual inspection of all 2,049 micrographs, a total of 207,410 particles were obtained. Two rounds of 2D classification were performed and bad particles were removed. The resulting data set of 134,512 particles was used for further refinement and focused classification refinement in 3D. The Bos taurus Pol II structure (EMDB accession code EMD-3219) (Bernecky et al., 2016a) was low-pass filtered to 40 Å as an initial model for 3D refinement. Initial 3D refinement followed by movie processing and particle polishing yielded a reconstruction at an overall resolution of 3.4 Å (gold-standard Fourier shell correlation criterion 0.143, RELION 2.1). Focused 3D classification without image alignment was performed on the α-amanitin binding pocket, the Pol II stalk (RPB4-RPB7) and upstream DNA, followed by global 3D refinement.
2.2.6 Model building and refinement
Model building was based on the previously published bovine Pol II structure (PDB accession code 5FLM)(Bernecky et al., 2016a). The model was manually fitted in COOT (Emsley et al., 2010). The α-amanitin molecule was taken from a Saccharomyces cerevisiae amanitin-bound Pol II structure (PDB accession code 2VUM)(Brueckner and Cramer, 2008). The α-amanitin molecule was rigid body fitted into the density. The structure was refined in real
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space with special restraints to the nucleic acids and α-amanitin using PHENIX (Torices and Muñoz-Pajares, 2015).
2.2.7 Transcription assay
Template DNA and RNA were mixed at a molar ratio of 1:1 and annealed as described (Brueckner et al., 2007). The template annealed DNA-RNA was mixed with Pol II-hGdown1 complex at a molar ratio of 1:2 and incubated at 28 ℃ for 10 min. Non-template DNA was added and incubated at 28 ℃ for an additional 10 min. The elongation complex was mixed with α-amanitin or buffer (control) at the same molar ratio used for the complex formation.
The sample was subsequently incubated on ice for 20 min. 100 μM UTP was added to both control and experimental reactions. The reaction was incubated in transcription buffer at 28
℃ and samples were taken at indicated time points. The reaction was stopped by adding stop buffer to the reaction. The product RNA was separated using a 20% denaturing urea polyacrylamide gel (300V) and visualized using a GE Typhoon FLA 9500 (GE Healthcare).
3 Results
3.1 Purification of Sus scrofa Pol II
Sus scrofa Pol II was purified essentially as described for the bovine Pol II preparation, except that pig thymus instead of bovine thymus was used (Method). Briefly, thymus was
homogenized, and the supernatant was filtered. After polyethyleneimine precipitation, Pol II was purified with a MacroPrepQ column, followed by ammonium sulfate precipitation and an affinity column with 8WG16 (αRPB1 CTD) antibody-coupled Sepharose, a UnoQ anion exchange column, and finally a Sephacryl S-300 HiLoad sizing column (Figure 1.3). The typical yield was 2~4 mg from ∼500 g of thymus. The fractions from UnoQ column were strictly selected to avoid pig Gdown1 contamination, then hGdown1 which was expressed and purified from E.coli was combined with Pol II and purified by gel filtration. Incubation of Pol II and hGdown1 on ice can form a stable Pol II-hGdown1 complex (Figure 1.4A).
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Figure 1.3: Purification of Sus. Scrofa Pol II from pig thymus and formation of Pol II-hGdown1 complex. A, chromatogram of UnoQ column, Pol II was concentrated by UnoQ with a high peak coming out within the elution gradient. B, SDS-PAGE of UnoQ fractions, the volume increases from left to right, the earlier Pol II fractions including pig Gdown1 were trashed. C, chromatogram of HiPrep Sephacryl S-300 column. The volume of the column is 120ml. Pol II comes out around 50ml. D, SDS-PAGE of the gel filtration fractions. hGdown1 was bound to Pol II stably after incubation.