Promoter independent Pol II assay

3/15/07 Last summer when I worked on purifying Pol II, I modified the procedure described below the horizontal line. The information below the line is still pertinent so I haven't deleted this information. The major change was scaling down the reaction to 4 ul so I could conserve on reagents when assaying for Pol II off the columns. A single reaction was done as follows:

1.35 ul of protein fraction was put in a 600 ul tube and 2.65 ul of reaction mix was added. The mixture was incubated at room temperature for 20 minutes. Draw a grid of 1 cm squares on a piece of DEAE paper. At the end of the transcription reaction, spot each sample in a separate square on the grid. Wash the paper in phosphate solution as described below. Cut the grid into separate squares and count the radioactivity in a scintillation counter.

2.65 ul of reaction mix contains the following (Scale up for the desired number of reactions. Excess can be stored in the freezer for at least 1 week.)

2 ul of 2X trancription mix minus nucs (2 mM MnCl₂, 50 mM HEPES pH 7.6, 24% glycerol, 100 mM (NH₄)₂SO₄, 1 mM DTT, 160 ug/ml denatured, sonicated salmon or calf DNA)
0.4 ul of 5 mM GTP, 5 mM ATP, 5 mM UTP, 0.01 mM CTP
0.15 ul of water or 266 ug/ml alpha amanitin
0.1 ul of ³²P-alpha CTP

Note that this reaction is being done with ³²P-alpha CTP and the assay described below is being done with ³²P-alpha UTP. Either works but you must choose the cold nucleotides so that the one corresponding to the hot nucleotide is the one whose concentration is low.

(6/23/03) This assay is for promoter-independent RNA polymerase II transcription. I've used it to track RNA polymerase II during purification from embryos and larvae. Denatured salmon sperm DNA is used as template. Transcription is monitored by measuring the incorporation of radioactive UTP into a form that binds DEAE paper. The conditions are derived from Sluder, Greenleaf (1985) JBC 262:3244 with the modification of Zehring, et al. (1988) PNAS 85:3698, which replaces Mg⁺⁺ with Mn⁺⁺.

Standard transcription assays contain (This does not list the additional components contributed by the RNA polymerase II. For example, the final HEPES concentration in the reaction includes the 25 mM HEPES listed below plus the HEPES contributed by the RNA polymerase II solution):

0.5 mM GTP
0.5 mM ATP
0.5 mM CTP
0.025 mM UTP (this can be reduced to 0.005 mM to increase the specific activity of the UTP and increase the sensitivity of the assay)
0.25 uCi ³²P-alpha UTP
1 mM MnCl₂
25 mM HEPES pH 7.6
12% Glycerol
50 mM (NH₄)₂SO₄
0.5 mM DTT
80 ug/ml denatured, sonicated salmon sperm or calf thymus DNA

I recommend preparing a 2X stock of the components described above minus ³²P-UTP. Store this stock at in the freezer. Add the ³²P-UTP to an appropriate amount of 2X stock just before needed.

The RNA polymerase II is in a buffer containing of 25 mM HEPES (pH 7.6), 0.1 mM EDTA, 15% glycerol, ~500 mM KCl, 1 mM DTT, 0.5 mM PMSF. Our preparations are eluted from a mono Q column. 2 ul of material should be sufficient to detect activity.

It is important to run an alpha amanitin control with alpha amanitin present at a final concentration of 5 ug/ml in order to ascertain whether the radioactivity that sticks to the DEAE paper is produced by RNA polymerase II.

A typical reaction might be set-up as follows.

6.5 ul of 2X buffer supplemented with radioactive UTP
1 ul of 62.5 ug/ml alpha amanitin (in water) or 1 ul of water
3.5 ul of 500 mM KCl, 25 mM HEPES (pH 7.6), 0.1 mM EDTA, 15% glycerol
2 ul of RNA polymerase II (add this last)

Assemble the components on ice in the order described and mix gently. Reactions are incubated at 25^oC for 20 minutes. Transcription activity is measured by determining the fraction of radioactivity that sticks to a small piece of DEAE filter paper after the filter paper has been washed with 5% K₂HPO₄, 0.1% Na pyrophosphate. For each sample cut a square of DEAE paper about 1 cm per side. With a pencil, number each filter. Spot 5 ul of the transcription reaction (after a 20 minute incubation) on the paper and count the paper in a scintillation counter. This represents total counts. Wash the filters for a total of 30 to 60 minutes with 4 changes of 5% K₂HPO₄, 0.1% Na pyrophosphate. Count the filters again - there is no need to dry the filters. This represents incorporated counts.