Non-specific (promoter independent) RNA polymerase I activity assay
Julio Saez-Vasquez and Craig S. Pikaard, Washington University at St. Louis
This protocol is used to detect RNA polymerases in plant cell-free extracts or chromatography fractions due to the ability of RNA polymerases to catalyze the incorporation of radioactive nucleotide triphosphates into RNA. To assay RNA polymerases II or III, simply leave out the alpha-amanitin.
References: The protocol below is a modification of:
1) Roeder, R.G. (1974). JBC 249:241-248.
2) Schwartz et. al. (Roeder lab). 1974. JBC 249:5889-5897.
A)Solutions needed:
a) 2X reaction stock mix (using alpha-labeled 32P-GTP as the radioactive label):
buffer composition recipe for 1ml
100mM HEPES-KOH, pH 7.9 100ul 1M stock
4mM MnCl2 (Manganese!) 4ul 1M stock
1mM UTP,(Sigma U1006) 10ul 100mM stock
1mM rATP, (not deoxy; Sigma A6559) 10ul 100mM stock
1mM rCTP (not deoxy; Sigma C8552) 10ul 100mM stock
0.08mM cold rGTP (Sigma G3776) 0.8ul 100mM stock
2mg/ml BSA (acetylated, Boehringer) 200ul 10mg/ml stock
10ug/ml a-amanitin (Sigma A2263) 10ul 1mg/ml stock
50ug/ml calf thymus template DNA 100ul 0.5 mg/ml stock
100mM KCl 50ul 2M stock
sterile water 505 ul
b) 0.5 M sodium phosphate, dibasic (Na2HPO4)
268 grams added very slowly to 2 liters Milli Q water that is vigorously stirring with a stir bar. Let each clump of crystals dissolve before adding more, or they clump up and never dissolve. This may take an hour to make.
Procedure
1) dialyze all fractions to be tested into CB100 (25mM HEPES, pH7.9, 20% glycerol, 100mM KCl, 1mM DTT, 0.1mM EDTA).
2)determine the number of assays to be run and make the hot mix:
A) aliquot to a new tube 20ul of 2X mix per reaction (for 10 assays, you will need 200ul)