Plant RNA Polymerase I Holoenzyme Purification
Saez-Vasquez and Pikaard (1997) PNAS 94: 11869-11874; Saez-Vasquez and Pikaard (2000) J. Biol. Chem. 275:37173-37180
You will need the following buffers :
Nuclei Resuspension Buffer (RB0): 50 mM Hepes, pH 7.9, 20% Glycerol, 10 mM EGTA, 10 mM MgSO4, 0.5% Triton X-100
Nuclei Resuspension Buffer (RB100): Resuspension buffer containing 100 mM KCl
Honda Buffer: 0.44 M sucrose, 1.25% ficoll, 2.5% dextran T40, 20 mM Hepes-KOH, pH 7.4, 10 mM MgCl
CB100: 20 mM Hepes-KOH, pH 7.9 20% Glycerol, 0.1 mM EDTA, 100 mM KCl
CB1000: 20 mM Hepes-KOH, pH 7.9, 20% Glycerol, 0.1 mM EDTA, 1000 mM (1M) KCl
**just before using buffers, add fresh DTT (to 0.5 mM)
**before homogenization step, add Protease Inhibitors to Honda buffer: 1 mM PMSF, 2.5 μg/ml antipain, 0.35 μg/ml bestatin, 0.5 μg/ml leupeptin, 4.0 μg/ml pepstatin
Bio-Rad Assay of column step elutions: 2 ml Bio-Rad concentrate + 7 ml water = 9 ml total. Add 90 μl/well of a microtiter dish , then add 10 μl of CNE (crude nuclear extract) or fractions to be tested
PURIFICATION PROTOCOL
Crude nuclear extract
ALL THE STEPS MUST BE DONE IN COLD ROOM or at 4 degrees C
1. Start with 75 to 100 grams of Broccoli inflorescence. Take only the clusters of rapidly dividing cells; avoid the stems. Use a razor blade.
2. Grind (6 pulses of 5 seconds each) in stainless steel Waring blendor containing 200 ml of Honda buffer (+DTT and protease inhibitors)
3. Filter through 2 layer of Miracloth
4. Spin at 7,500 rpm, 30 minutes in Beckman rotor JA10, 4 degrees C, to obtain a crude nuclear pellet
5. Resuspended pellet in 20 mL of RB0+DTT (0.5 mM)+PMSF (1 mM)
6. Add 1.16 grams of NaCl and mix by hand, incubate 10 minutes
7. Add 0.4 ml of PEG8000-50% (1/50, original protocol), mix gently w/magnetic bar for 20 minutes
8. Spin at 12,000 rpm, 30 minutes, JA20 rotor (or 11,000 rpm in JA14 if the volume is large), 4 degrees C
9. Recover the supernatant and spin it again at 12,000 rpm, 30 minutes, JA20 (or 11,000 rpm, JA14), 4 degrees C.
10. Recover the supernatant and dilute with RB0 to 100 ml
(you will see viscous chromatin floating in the diluted supernatant)
11. Filter through 2 layers of Miracloth (do it twice if required).
12. Recover the supernatant and precipitate with 0.33 g/ml ammonium sulfate NH4(SO4), (Add NH4(SO4) very slowly over a period of 20-30 minutes
then, stir for approximatly 1 hr.)
13. Spin at 12,000 rpm, 30 minutes, JA20 (or 11,000 rpm, JA14), 4 degrees C.
14. After spinning you will see that the pellet forms along the walls of the centrifugation bottle. Very carefully discard supernatant-the pellet is loose.
15. Resuspend pellet in ≈12.5 ml of RB100+DTT and proteases inhibitors (PMSF, antipain, bestatin, leupeptin, antipain A) using a Dounce homogenizer
16. Spin at 12,000 rpm, 30 minutes, JA20 rotor, 4 degrees C
Dialyze against RB100+DTT (0.5 mM)+PMSF (1mM) (at least 3 changes of 500 mL each)
Spin at 12,000 rpm, 30 minutes, JA20, 4 degrees C
Store at -800C or begin chromatography procedures.
References for cell-free extract procedures
Chris Lamb Lab., Plant Journal 7(6), 1021-1030 (1995) Ethan Signer Lab., Plant Mol. Biol. 18: 865-871 (1992)
4-column purification of the Pol I holoenzyme
DEAE-Sepharose Cl-6B
Approximately 12.5 ml of crude nuclear extract are loaded onto ≈ 10 ml DEAE-sepharose Cl-6B 9 (small column ≈ 2.5 cm diameter)
Wash the column with 5 volumes of RB100 (100 mM KCl)
Wash the column with 5 volumes of RB175 (175 mM KCl)
Elute RNA pol I holoenzyme fraction with RB400 (400 mM KCl)
Collect ≈ 4-5 ml fractions and test for proteins by mixing 10 ul of each fraction with protein reaction mix (70 ul H20 + 20 ul Bradford reagent). The degree to which sample turns blue provides a visual way to follow the peaks eluting from the column and allows you to know when you have reached baseline during the washes.
Note: If making 10 extracts on one day, use a 100 ml DEAE sepharose Cl-6B column (big column ≈ 5.5 cm diameter)
Biorex70
Approximately 12 ml of fraction DEAE-F400 are loaded onto ≈ 2 ml Biorex 70 column (small column ≈ 1.5 cm diameter)
Wash with 10 ml RB100
Elute holoenzyme RNA pol I fraction with 10 ml of RB800 (800 mM KCl)
Collect ≈ 1 ml fractions and test for protein by mixing 10 ul of each fraccion with protein reaction mix (70 ul H20 + 20 ul Bradford reagent)
Note: If making 10 extracts at once, use a 10 ml Biorex (big column ≈ 2.5 cm diameter) Sephacryl S300
Approximatly 3 ml of Biorex F800 fraction is immediately loaded onto ≈ 190 ml Sephacryl S300 FPLC column equilibrated in CB100
FPLC program:
Flowrate:1 ml/min
Speed of Chart: 0.25 cm/ml Collector: 1 ml/min
Start collecting at 60 ml Stop collecting at 120 ml End of the program
Peak fraction should elute between fractions 10 to 20 (≈70 to ≈80 ml); due to desalting effect on the gel filtration column, these RNA Pol I fractions are now in CB100.
MonoQ
Approximatly 10 ml of sephacryl fraction are immediately loaded onto a 2 ml MonoQ FPLC column equilibrated on CB100
FPLC program:
At 0 ml
Flow rate 1 ml/min (0.8 ml/min) Chart speed 1 cm/min
Collector 1.25 ml/min
At 5 ml alarm goes ON
Start injection
At 15 ml alarm goes OFF
At 25 ml beginning of the gradient Start collecting
At 45 ml end of gradient (600 ml KCl) Wash at 1000 mM KCl
At 5 ml stop Wash
Stop collecting
Salt goes down to 100 mM
Program stop at 55 ml
Collect fraction from 10 to 20 for dialysis against RB100 (0.5 mM DTT). Holoenzyme RNA pol I should be between fraction 13 to 16. Verify by the assay of RNA pol I non-specific activity, promoter dependent activity and/or western blot using antibodies against LE-RNA pol I (largest RNA pol I subunit, last exon)