Analisi di terminazione della trascrizione Utilizzando BrUTP-strand-specifici Trascrizione Run-on (TRO) Approccio

The overall goal of this procedure is to study the termination of transcription by RNA polymerase to in budding yeast under in vivo conditions by the strand specific transcription run on approach. This method helps answer the key question of whether the transcriptionally active polymerase detected beyond the three prime end of the gene is the one initiated from the promoter region. The main advantage of this method is that it can distinguish an MRNA transcribing polymerase from a pervasively transcribing polymerase that initiated transcriptions and will be on the three prime end of the gene.

This technique can be extended to study the termination of transcription by other RNA polymerases and yeast due to the conserve nature of termination by different polymerases. Though this method can provide insight into the precise role of termination factors in the yeast transcription cycle it can also be extended to study termination in higher eukaryotes. Generally, individuals new to this method will struggle with cell permeabilization and transcription run on stops, as they require careful execution, and nascent transcripts can also be difficult to purify.

We first had the idea for this method from the GRO seek approach that gives a genome wide snapshot of the position of transcriptionally active polymerases in a strand specific manner. Visual demonstration of this method is critical, as the transcription run and RNA purification are difficult to perform due to the low amount and unstable nature of the nascent transcripts. Prior to starting the transcription run on reaction, grow, harvest, and permeabilize exponentially growing yeast cells, to obtain a permeabilized cell pallette.

To start the reaction add 150 microliters of transcription run on buffer to the permeabilized cell palette. Using a truncated P-200 tip mix gently by pipetting up and down. Place the sample on ice.

After all samples have been resuspended in the transcription run on buffer, incubate the samples for five minutes at 30 degrees Celsius in a water bath. Stop the reaction by adding 500 microliters of cold ready to use RNA isolation reagent to each sample. Incubate for five minutes at room temperature.

Begin the RNA extraction procedure by adding 200 microliters of acid washed glass beads to each cell suspension and shaking vigorously in a vortex mixer for 20 minutes at four degrees Celsius. After 20 minutes, use a hot 22 gauge needle to puncture the bottom of each microcentrifuge tube and place the tube on the top of a 15 milliliter sterile conical tube. Centrifuge at 300 times G for one minute at four degrees Celsius and transfer the cell lysate to a 1.5 milliliter microcentrifuge tube.

Add 500 microliters of cold RNA isolation reagent, and 200 microliters of chloroform to the cell lysate. Mix the contents on the vortex mixer and then centrifuge at 16, 168 times G for 20 minutes at four degrees Celsius. Transfer the upper aqueous phase to a new microcentrifuge tube.

Add an equal volume of cold phenol chloroform isoamyl alcohol. Shake vigorously on the vortex mixer and then centrifuge at 16, 168 times G for 15 minutes at four degrees Celsius. Transfer the upper aqueous phase containing RNA to a new microcentrifuge tube.

Add an equal volume of cold phenol chloroform isoamyl alcohol, vortex and centrifuge again. To the final RNA containing aqueous phase add an appropriate volume of a five molar sodium chloride stock to get a final concentration of 0.3 molar. Add three times the volume of cold ethanol to precipitate the RNA.

Incubate for one hour, or overnight, at 20 degrees Celsius. When the RNA precipitation is done, centrifuge at 16, 168 times G for 20 minutes at four degrees Celsius. Remove the supernatant and resuspend the RNA palette in 100 microliters of DEPC water.

Use a RNA isolation kit to separate the RNA from the unincorporated bromouridine triphosphate nucleotides and elute the RNA from the column with 100 microliters of RNAse free water. Incubate the RNA in a 65 degree Celsius water bath for five minutes. And then transfer to ice for at least two minutes.

Start this procedure by preparing the anti bromo DU beads while the RNA is being precipitated. For each sample add 500 microliters of 0.25 X sodium saline phosphate EDTA binding buffer to 25 microliters of settled beads. Centrifuge at 200 times G for 30 seconds at four degrees Celsius, and remove the supernatant.

Wash the beads three times with the binding buffer. After removing the supernatant from the third wash, add 500 microliters of blocking buffer to the beads and shake gently for one to two hours at four degrees Celsius on a platform shaker. Wash the beads two more times with 500 microliters of binding buffer.

Remove the supernatant from the second wash and add 400 microliters of binding buffer to the beads. Transfer 100 microliters of RNA directly to the beads. Incubate with gentle shaking for one to two hours at four degrees Celsius on a platform shaker.

Spin at 200 times G for 30 seconds at four degrees Celsius and then remove the supernatant. Next, add 500 microliters of binding buffer to the beads. Spin at 200 times G for 30 seconds at four degrees Celsius and remove the supernatant.

In this manner, wash the beads sequentially with binding buffer, low salt buffer, high salt buffer, and twice with TET buffer. To elute the bromouridine triphosphate labeled RNA from the beads add 150 microliters of elution buffer and incubate for four minutes at 42 degrees Celsius in a water bath. Spin down briefly and collect the supernatant.

In this manner, elute sequentially twice with 150 microliters and once with 200 microliters of elution buffer. About 500 microliters of affinity purified bromouridine triphosphate labeled RNA should be obtained from each sample. To precipitate the affinity purified bromouridine triphosphate labeled RNA add 500 microliters of cold phenol chloroform isoamyl alcohol to each sample.

Vortex and centrifuge at 16, 168 times G for 15 minutes at four degrees Celsius. Transfer the upper aqueous phase to a new microcentrifuge tube. Add the appropriate volume of a five molar sodium chloride stock to get a final concentration of 0.3 molar.

Then add three times the volume of cold ethanol. Incubate at 20 degrees Celsius overnight to precipitate the RNA. On the following day, collect the precipitated RNA by centrifugation at 16, 168 times G for 30 minutes at four degrees Celsius.

Remove the supernatant and allow the palette to air dry for 10 minutes. Resuspend the RNA palette in 26 microliters of DEPC water. Determine the final RNA concentration by measuring the absorbance at 260 nanometers using a spectrophotometer.

Store the RNA samples and aliquots at 80 degrees Celsius until needed for cDNA synthesis. The bromouridine triphosphate strand specific transcription run on approach was used to test the requirement of RNA 14 for termination of transcription of the ASC1 gene in budding yeast. Bromouridine triphosphate labeled RNA from the RNA 14-1 mutant and its isogenic wild type cells was purified and reverse transcribed with primers C, D, E, F, and G.The corresponding cDNA was PCR amplified using primer pairs B and C, B and D, B and E, B and F, and B and G, and fractionated on agarose gels.

The presence of PCR amplified products from the primer pairs B and E, B and F, and B and G in the mutant, reflects a termination readthrough phenotype. In the wild type cells however the transcription run on signal was limited til the terminator element and there was no detectable transcription run on signal beyond the terminator region. This graph shows the quantification of the gels with five S RNA as the normalization control.

The detection of promoter initiated nascent transcripts that read through the termination signal in the mutant but not in the wild type cells confirms that RNA 14 is a termination factor. Once mastered, this technique can be done in three days. While attempting this procedure it is important to remember that the transcription run on reaction is critical for getting a successful result.

Following this procedure other methods such as GRO seek can be performed in order to answer additional questions like the genome wide role of a factor in termination. After its development this technique paved the way for researchers in the field of transcription to explore termination by other RNA polymerases in yeast and higher eukaryotic systems. After watching this video you should have a good understanding of how to perform transcription run on reactions and purify nascent labeled transcripts.

Don't forget that the RNA can be extremely unstable and precautions such as wearing gloves, keeping the temperature around four degrees Celsius, and using RNAse free agents should always be taken while performing this procedure.