Protein-tRNA Agarose Gel Retardation Assays for the Analysis of the N⁶-threonylcarbamoyladenosine TcdA Function

The overall goal of this procedure is to demonstrate an RNA gel retardation assay designed for the discovery and characterization of RNA-binding proteins. Application of this assay is used to investigate the interaction between TcdA and transfer RNA. The agarose gel retardation method can provide answers to key questions in the field of RNA biology.

For example, whether a certain protein interacts with RNA, whether the interaction is sequences-specific, and the strength of the interaction. The main advantages of this technique are that it uses various small amounts of protein and RNA samples, and yields very informative results about the protein-RNA interactions quite quickly. Though this method can provide insight into protein-tRNA complexes, it can also be applied to many other systems such as ribosomal RNA, messenger RNA, and any other kinds of RNA molecule.

In the morning, retrieve a vial of frozen E coli BL21 DE3 transformed with the expression vector pET-23D EC triple-U. Streak a low salt luria broth or LB plate supplemented with 100 micrograms per milliliter ampicillin. Invert the plate and place it into a 37 degrees Celsius incubator until colonies appear in the late afternoon.

Inoculate one well-grown single colony in five milliliters of LB plus 100 micrograms per milliliter of ampicillin and grow overnight. The next morning, pellet the cells. Following centrifugation, replace the medium with five milliliters of fresh LB plus 100 micrograms per milliliter of ampicillin and re-suspend.

Use this suspension to inoculate 200 milliliter of LB plus 100 micrograms per milliliter of ampicillin. Grow the cells for five hours at 220 RPM and 37 degrees Celsius. When the optical density of the culture at 590 nanometers has reached 0.6 to 0.8, atdd1 millimolar IPTG to the culture to trigger the expression of the tRNA triple-U gene.

After incubating the cells at 37 degrees Celsius for three hours, harvest the cells by centrifuging the culture at 6500 times g for 10 minutes at four degrees Celsius. To perform lysis, first re-suspend the cell pellet in five milliliters of lysis buffer. Add one volume of acid phenol to the re-suspended cell pellet in the fume hood and mix for one minute by inversion to lyse the cells.

Then, centrifuge at 10 thousand times g for 10 minutes at 4 degrees Celsius. Collect the upper aqueous phase containing the soluble tRNA triple-U using a pipette. Take care not to aspirate the organic bottom phase.

Precipitate the tRNA by thoroughly mixing the soluble phase with 2.5 volumes of 100%volume per volume ethanol by inversion for one hour at four degrees Celsius. Then, centrifuge at 15 thousand times g for 30 minutes at four degrees Celsius. Decant the supernatant carefully, then resuspend the tRNA-rich pellet in four milliliters of gel filtration buffer.

Following standard chromatographic practice, load the re-suspended tRNA triple-U onto a high-resolution preparative gel filtration column previously equilibrated in gel filtration buffer. Set the flow rate to 1 milliliter per minute to elute the samples. To begin sample preparation, pipette the corresponding amount of TcdA as indicated in the text protocol into properly labeled 1.5 milliliter centrifuge tubes.

Add a final concentration of 1.6 millimolar TCEP and gently mix using a pipette. Close the lid and incubate the mixture at room temperature for five minutes. Following incubation, add adenosine triphosphate and magnesium chloride.

Mix with a pipette before incubating the mixture for five minutes at room temperature. Next, add purified tRNA triple-U according to the text protocol and mix the sample with a pipette. After incubating the sample at room temperature for five minutes, add phosphate buffered saline up to a 100 microliter final volume.

Mix properly and add glycerol to a final concentration of 10%Vortex the sample before briefly spinning down the contents of the tube. Prepare the agarose gel as described in the text protocol. Place the electrophoresis unit into a tray filled with crushed ice to maintain the chamber at four degrees Celsius during the electrophoresis run.

Carefully pipette five microliters of each sample into the corresponding well. Also add a suitable DNA size marker. Then, plug the electrodes into the corresponding slots of the power supply unit.

After turning on the power supply unit, set the voltage to 100 volts and run the gel for 90 minutes. Next, prepare the staining solution by mixing 50 milliliters of TBE with 1X of a suitable DNA stain. After 90 minutes of electrophoresis, carefully remove the gel from the tray and place it in a container with staining solution.

Place the container on a rocker at low rocking speed and rock at room temperature for 30 minutes. Remove the agarose gel from the staining container and tap it carefully on a piece of cellulose paper to remove excess liquid. Then, place the gel directly on a transilluminator.

Switch on the UV light to visualize bands containing tRNA. Finally, take a photograph of the gel. Shown here are representative results of tRNA purification for gel retardation assays.

In the chromatogram the absorbance of the elution fractions at 254 nanometers is recorded. The tRNA pool, enriched in over-expressed tRNA triple-U elutes at the highest peak. The quality and quantity of the tRNA pool is examined by agarose gel electrophoresis.

The RNA gel retardation assay reveals that TcdA forms a stable complex with tRNA triple-U in tRNA concentration-dependent manner, which results in a protein-tRNA band that migrates more slowly than the free tRNA at the front of the gel. Once mastered, this technique can be done in less than two hours. While attempting this protocol, it's important to remember to follow strict laboratory techniques to ensure the chemical stability of tRNA.

After watching this video, you should have a good understanding how to use agarose gel retardation assay to investigate the interaction of your protein of choice with tRNA, as in the case of TcdA or with any other type of RNA.