The overall goal of this in vitro transcription assay is to study the complex regulatory steps during transcription by providing a platform for testing the effects of transcription factors, small molecules, and transcription inhibitors. This method can help answer key questions in molecular biology, such as the precise mechanisms associated with transcription regulation, the function of transcription factors with unknown mechanisms, and the effects of normal transcription inhibitors. The main advantage of this technique is that it allows direct visualization of all the transcription products along a timeline from a single RNA sequencing gel.
Begin this procedure by cleaning the plates from a sequencing gel apparatus twice with 70%ethanol and purified water. Assemble the cell with a comb and two spacers. Dissolve 33.6 grams of urea in 22 milliliters of water and 16.4 milliliters of five times TBE buffer using a magnetic stirrer.
Add 16 milliliters of a 40%acrylamide, this acrylamide solution to the urea solution, and filter through a 0.45 micrometer filter. Add to the filtered solutoin 514 microliters of a freshly prepared 10%ammonium persulfate solution. Followed by 51.4 microliters of TEMED.
Gently invert the gel solution to avoid excessive aeration and inject immediately and smoothly from the bottom injection hole into the horizontally positioned pre-packed sequencing cell. Take care to avoid making bubbles during the injection. Allow the gel to completely set for one to 1.5 hours before use.
To start the procedure for the formation of a transcription elongation complex, add one microliter of RNA polymerase core enzyme to a 1.5 microliter tube. Add one microliter of purified sigma 70 factor to the RNA polymerase and incubate on ice for 10 minutes to form the RNA polymerase holoenzyme. Add to the RNA polymerase holoenzyme 3 microliters of a 500 nano molar purified template DNA in transcription buffer and incubate at 37 degrees Celsius for five minutes to form the open complex.
Next, prepare a mixture of reaction components by adding the following reagents to a 1.5 milliliter tube. Five microliters of 10 times transcription buffer, two microliters of 250 micromolar GTP, two microliters of 250 micromolar UTP, one microliter of one millimolar ATP, and one microliter of alpha P 32 labeled UTP. Add DEPC treated water to the mixture to 41 microliters.
Transfer the mixture to the tube containing the open complex and incubate at 37 degrees Celsius for fifteen minutes to form the transcription elongation complex stopped at plus 26 nucleotide. After 15 minutes, move the reaction to room temperature and add one microliter of 2.4 milligrams per milliliter rifampicin to a final concentration of 50 micrograms per milliliter. Add one microliter of 0.1 millimolar purified NusA transcription factor to the reaction mixture and incubate for five minutes at room temperature.
Add two microliters of 250 micromolars CTP to the reaction mixture to make a final volume of 50 microliters and measure the reaction time using a timer. At each time point, transfer five microliters of the reaction solution into 2 microliters of RNA Gel-loading Buffer to quench the reaction. Prior to electrophoresis of the samples, pre-run the gel for approximately 1.5 hours at 50 degrees Celsius and 50 watts in one time TBE buffer until the temperature of the gel and the buffer is stable at 50 degrees Celsius.
When the pre-run is complete, heat the samples at 90 degrees Celsius for 30 seconds and move them onto ice. Load the first well on the top of the gel with two microliters of RNA ladder and then, load the samples into the wells along side the ladder. Run the gel at 50 watts and 50 degrees Celsius in one time TBE buffer for about 1.5 hours, until the bromophenol blue dye reaches about 2/3 down the gel.
When the run is complete, slowly open the two gel plates and carefully separate the upper plate from the sequencing cell, taking great care to avoid breaking the gel. Cut chromatography paper to match the gel size from the top to the bromophenol blue dye. Use a Geiger counter to confirm the approximate position of radioactively labeled RNA transcripts.
Carefully cover the gel with the filter paper and press firmly until the gel adheres to the filter paper. Cut off and discard the bottom of the gel as radioactive waste, since the unincorporated NTPs run to the bottom of the gel, which can be highly radioactive. Place a similar sized piece of filter paper on the drying bed of a vacuum drying machine and carefully lay the gel-coated paper onto it with the gel side up.
Cover the gel with plastic wrap and dry at 60 degrees Celsius for 1.5 hours. After the gel is dry, wrap the other side of the gel-coated filter paper with plastic wrap and place a photostimulable phosphor plate in the imaging case for the appropriate exposure time. Subsequently, image the phosphor plate using an imager scanner according to the manufacturer's instructions.
To test the effects of mutant NusA end terminal domain, or NTD in transcription, transcription elongation pausing assays were performed without NusA or with NusA-NTD, NusA-NTD with Helix 3 deleted, and NusA-NTD with alanine substitutions at eight residues. The pause, termination, and run-off products were successfully visualized. In the presence of NusA-NTD, appearance of the RNA products was significantly delayed compared to the control experiment lacking NusA.
The pause activities of half-lives of mutant NusA-NTD relative to the wild type protein is shown. Deletion of helix 3, or alternation to alanine of a series of amino acids, resulted in the complete loss of NusA pause activity. Helix 3 residues, arginine 104 and lysine 111, were shown to be essential for NusA-NTD pause activity.
In vitro transcription assays can also be used to examine the effects of newly identified bacterial transcription inhibitors, such as compound C5.Percent inhibition of transcription was determined by the RNA product relative to a control against the concentration of C5 used in the reaction. Mechanistically, addition of C5 to RNA polymerase followed by sigma 70 factor to the reaction mixture was significantly more efficient for transcription inhibition than using C5 after the formation of the RNA polymerase holoenzyme. Following this procedure, other methods like ELISA and isothermal titration calorimetry, can be preformed in order to answer additional questions, such as, how a inhibitor affects transcription.
Once developed, this technique pave the way for researchers in the field of molecular or chemical biology. To explore the mechanism and related inhibitors in bacterial transcription. Don't forget that working with radioactive reagents can be extremely hazardous and precautions such as full PPE should always be used while preforming this procedure.
