The overall goal of this protocol is to biochemically characterize tRNA-Isopentenyl Transferase Activity In Vitro. This method is useful for the in-vitro detection and characterization of tRNA-isopentenyl transferase activity of purified recombinant enzymes. The main advantage of this technique is that it is a simple and direct assay used to detect a covalent RNA modification.
Though this method provides insight to tRNA-isopentenyl transferase activity, it's potentially adaptable for the biochemical characterization of wide range or RNA modifying enzymes. The first step in this procedure is to thoroughly clean the glass plates that will be used for casting the gel. Wash the plates with soap and water, rinse well with deionized water, and then clean the plates with isopropanol and lint-free wipes.
Next, assemble the plates and spacers. Mix the following reagents to make a 100 milliliter 20%acrylamide, 7.5 molar urea, 1x TBE gel. 80 milliliters of urea gel concentrate, 10 milliliters of urea gel diluent, and 10 milliliters of urea gel buffer.
Add 40 microliters of T-med and 800 microliters of freshly prepared 10%ammonium persulfate to the mixture. Draw the gel solution up into a large syringe and dispense the gel solution between the glass plates tapping on the glass while dispensing the solution to prevent bubble formation. Allow the gel to solidify at room temperature for 30 minutes.
After the gel has solidified, plant the gel onto a vertical gel apparatus. Fill the upper and lower buffer chambers with 1x TBE. Two hours prior to loading the gel, pre-run the gel at 20 milliamps for two hours to allow the buffer boundary to out run the smallest oligonucleotides.
Prepare each reaction for the RNA isopentenylation assay in a final volume of 17 microliters. Add to each tube 50 millimolor Tris-HCl, pH 7.2, 1.2 millimolar ATP, 5.8 millimolar magnesium chloride, 0.2 millimolar DMAPP, 10 units RNase inhibitor, 40, 000 CPM internally 32P labeled RNA, 5.3 micromolar Mod5, and 1.2 millimolar 2-mercaptoethanol. Incubate the reactions at 37 degrees Celsius for one hour.
After one hour, ethanol precipitate the RNase using 2.5 volume of 100%ethanol and 1/10 volume of 3.5 molar sodium acetate pH 5.5, and place it negative 20 degrees Celsius for one hour overnight. Next, centrifuge the RNA samples at 15, 400 g's and four degrees Celsius for 20 minutes. Carefully remove the supernatant and wash each RNA pellet with 500 microliters of 70%ethanol.
Centrifuge the samples at 15, 400 g's and four degrees Celsius for five minutes. Carefully remove the supernatant and air dry the RNA pellets for 15 minutes or until all ethanol has evaporated. Next re-suspend each RNA pellet in 10 microliters of eight molar urea.
Add 150 units of RNase T1 to each sample and incubate at 37 degrees Celsius overnight. On the following day, add two microliters of 6x loading buffer to each sample. Load 10 microliters of each RNA sample on a pre-run, 20%polyacrylamide, 7.5 molar urea gel.
Run the gel at 25 milliamps for two hours. After two hours, stop the electrophoresis and remove the gel from the apparatus. Break the seal between the two glass plates and carefully remove one of the glass plates with the gel remaining on either plate.
Place a layer of plastic wrap over the gel and expose on a phosphorus screen for three hours. This protocol reliably detects isopentenylation of both canonical and noncanonical tRNA residues modified by the S.cerevisiae isopentenyl tranferase Mod5. In this example tyrosine tRNA was internally labeled with 32P-adenosine and Mod5 was incubated with RNase in the presence or absence of DMAPP.
The RNAs were digested with RNase T1 and resolved on a denaturing polyacrylamide gel. Mod5 modifies the predicted residue in the presence of DMAPP and indicated by the shifted 10 nucleotide, triple adenosine containing fragment at nucleoside positions 36 through 38 contain fragment in the tyrosine tRNA. The modified adenosine 37 reside is indicated with an asterisk.
Similarly when the serine tRNA is incubated with Mod5 and DMAPP, a complete shift of the 10 nucleotide triple adenosine containing fragment at nucleoside position 36 through 38 is observed. Interestingly, a partial shift of a seven nucleotide fragment is observed that does not contain the triple adenosine containing fragment at nucleoside positions 36 through 38, suggesting that the triple adenosine containing fragment at nucleoside position 36 through 38 sequence and structure may not be required for Mod5 in vitro activity. Once mastered this technique will take two, four to six hour days if it is performed properly.
Well attempting this procedure it is important to remember to maintain and monitor the integrity of the RNA, because RNA degradation could effect the RNA modification and will confound data interpretation. Following this procedure mutational studies using your enzyme of interest, could be done to determine specific residues or domains required for enzymatic activity. After its development, this technique paved the way for researchers in the field of RNA biology, to say tRNA-isopentenyl transferase activities of prokaryotic and eukaryotic enzymes.
After watching this video, you should have a good understanding of how to carryout the protocol for the detection of isopentenyl transferase activity of your enzyme of interest. Don't forget that working with radioactivity can be extremely hazardous and precautions such as wearing proper PPE, using adequate shielding, and disposing of radioactivity properly should always be taken while performing this procedure.
