Here, we've established a simple experimental system that allows reconstitution of active Pol II elongation complexes suitable for investigating the functional coupling of Pol II transcription and RNA capping. With this method, functional elongation complexes can be easily assembled with just a few components. The elongation complexes are immobilized on magnetic beads, so it's easy to change reaction conditions.
To immobilize DNA oligo containing biotin on magnetic beads, first add 200 microliters of magnetic beads to a low-protein binding 1.5-milliliter tube, and then place the tube on a magnetic rack for two minutes. While the tube is on the magnetic rack, use a pipette to remove the liquid from the tube without disturbing the beads. To wash the beads, remove the tube from the rack, and add one milliliter of wash buffer containing Tris-hydrochloride, EDTA, and sodium chloride.
Return the tube to the rack. After two minutes, remove the wash solution, and repeat the washes two more times using 200 microliters of the same buffer. Resuspend the magnetic beads in 400 microliters of 2X buffer.
Then, add 380 microliters of water and 20 microliters of 10-micromolar non-template biotinylated DNA oligo to mix. Place the tube on a nutator, and incubate for 30 minutes at room temperature. After that, wash the beads with immobilized non-template DNA oligo three times using 200 microliters of buffer and then three times with 200 microliters of storage and dilution buffer.
Store the tube at four degrees Celsius overnight to finish blocking the beads with BSA. The next day, discard the solution, resuspend the beads in 200 microliters of storage and dilution buffer, to reach an approximate DNA concentration of five micromolar, and then transfer the solution with the beads to a new tube. To obtain the DNA-RNA duplex, prepare a 10-microliter annealing mix in a PCR tube with a two-to-one RNA-DNA molar ratio, as described in the written protocol.
Place the tube in a thermal cycler. Set the program to 45 degrees Celsius for five minutes, followed by 12 cycles of two minutes each, starting at 43 degrees Celsius and decreasing the temperature by two degrees Celsius per cycle. Lastly, keep it idle at four degrees Celsius.
To load purified RNA polymerase II onto the DNA-RNA hybrid, mix one microliter of DNA-RNA duplex with 13 microliters of freshly prepared polymerase II buffer in a tube. Add one microliter of purified RNA polymerase II, and mix by gently pipetting up and down and stirring with the pipette tip, without introducing bubbles. Incubate the tube for 10 minutes at 30 degrees Celsius.
To complete the elongation complex, add one microliter of immobilized non-template DNA oligo beads to 14 microliters of non-template DNA buffer, add the 15-microliter mixture to the previously incubated tube, and incubate it for another 10 minutes at 37 degrees Celsius. Place the sample on the magnetic rack for two minutes. Wash the sample one time with 30 microliters of wash buffer to remove unincorporated polymerase II and oligos.
To radiolabel the elongation complexes with RNA 23mers, add one microliter of ATP and one microliter of radiolabeled UTP to 23 microliters of pulse buffer, and add this mixture to the washed magnetic beads to resuspend. Incubate the reaction for 10 minutes to allow the synthesis of radiolabeled 23mers. Add 1.5 microliters of solution containing 100-micromolar ATP and 100-micromolar UTP mix to 3.5 microliters of chase buffer, and add the mixture to the tube containing preassembled elongation complexes on beads.
Again, incubate the tube for five minutes to chase all nascent transcripts into 23mers. Then, place the tube onto the magnetic rack, and wash as was done previously to remove unincorporated nucleotides. Resuspend the sample in 30 microliters of wash buffer.
To generate elongation complexes with longer transcripts, follow the previous procedures, and scale up four-fold to generate 120 microliters of washed elongation complexes for four reactions. Label three new tubes 23mer, 25mer, and 29mer. Transfer 30 microliters of washed elongation complexes to the 23mer tube, and add 94 microliters of stop mix containing proteinase K and glycogen.
Place the tube containing the remaining 90 microliters of washed elongation complexes on the magnetic rack for two minutes, remove the supernatant, and resuspend beads in 90 microliters of BTB supplemented with 1.2 microliters of ATP and CTP. Incubate for 10 minutes at 30 degrees Celsius. After washing with 90 microliters of wash buffer, transfer 30 microliters of elongation complexes to the 25mer tube, and add 94 microliters of stop mix containing proteinase K and glycogen.
Again, place the tube containing the remaining 60 microliters of elongation complexes on the magnetic rack for two minutes. Repeat the BTB treatment with 60 microliters of buffer supplemented with 0.8 microliters of ATP and CTP. After incubation and washing as done before, resuspend the sample in 60 microliters of wash buffer.
Transfer 30 microliters from the sample to the 29mer tube, and add 94 microliters of stop mix containing proteinase K and glycogen. Analyze reaction products on a denaturing polyacrylamide gel. First, generate washed elongation complexes containing 23mers by scaling up 13-fold the procedure previously described to obtain 390 microliters for 12 reactions and one extra.
To perform phosphorylation of polymerase II CTD, remove the supernatant after placing the sample in the magnetic rack, and resuspend the beads in 377 microliters of BTB. Label two new tubes plus H and minus H.To plus H, add three microliters of transcription initiation factor TFIIH at the concentration of 300 nanograms per microliter, and to minus H, add nine microliters of storage and dilution buffer. Add 87 microliters of the washed elongation complexes to the plus H tube and 261 microliters to the minus H tube.
Incubate the tubes for 10 minutes, and wash with wash buffer. To perform RNA capping, add 87 microliters of capping mix to the plus H tube and 261 microliters to the minus H tube to resuspend. Label four new tubes, five plus H, five minus H, 15 minus H, and 45 minus H, and dispense three microliters of five, 15, or 45 nanograms per microliter capping enzyme into the corresponding tubes.
Then, prepare 12 new tubes with 94 microliters of stop buffer. Start the first capping reaction by adding 87 microliters of elongation complex treated with TFIIH to the tube labeled five plus H.Incubate at 30 degrees Celsius in a heat block. The capping reactions need to be precisely timed using a stopwatch.
When working with multiple reactions, the start times for each reaction should be staggered. After one, two, or four minutes, transfer 30 microliters of the five plus reaction mixture to tubes labeled one, two, and three containing 94 microliters of stop buffer to stop the reactions. Proceed with capping reactions for the samples in the remaining minus H tubes in exactly the same way, using elongation complexes treated without TFIIH.
Analyze reaction products on a denaturing polyacrylamide gel. A diagram is shown here for DNA and RNA molecules in artificial elongation complexes. Using the synthetic RNA oligo of 20 nucleotides as the RNA primer during assembly of the artificial elongation complexes, nucleotides were added during each sequential walking step of the protocol to generate a 23mer, a 25mer, and a 29mer.
Reactions using RNA polymerase II from different sources were compared. Artificial elongation complexes were assembled with endogenous, wild-type polymerase II purified from either rat liver or fission yeast and walked to generate 23mers or 25mers. An assay was performed to measure capping of radiolabeled transcripts associated with artificial elongation complexes containing 23 nucleotide transcripts with a five-prime triphosphate end.
The last two lanes show the products of reactions in which elongation complexes were incubated with or without capping enzyme in the presence of TFIIH. The more slowly and more rapidly migrating bands contain capped and uncapped transcripts, respectively. The first 12 lanes show capping of transcripts associated with elongation complexes with either a phosphorylated or unphosphorylated CTD.
Activities of polymerase II and other enzymes may vary from prep to prep, so it's necessary to optimize reaction conditions by titrating enzyme concentrations to define optimal levels. It's important to use radioactive material safely and to avoid contamination. Please make sure to follow your laboratory and institute guidelines when doing experiments with radioactivity.
