Reporter Gene Repression Assay to Study Translational Regulation of a Target Gene

RNA-binding proteins, RBPs, function as post-transcriptional regulators by binding to the translation initiation region, TIR, of mRNA and repressing translation.

To study RNA-RBP interactions via translational repression of a reporter gene, take a bacterial cell culture containing two plasmids — the binding site and RBP plasmids.

The binding-site plasmid encodes a fluorescent reporter gene. Upon transcription, the resulting mRNA's TIR contains a ribosome-binding site and a hairpin structure, with a sequence recognized by RBPs.

Ribosomes bind to the binding site on the mRNA and destabilize the hairpin structure, leading to mRNA translation and fluorescent reporter protein formation. The RBP plasmid expresses a chimeric RBP fused to a different fluorescent protein. An inducible promoter controls the chimeric protein expression.

Add the inducer, which activates a transcription factor that binds to the promoter, inducing chimeric protein expression. The chimeric protein's RBP binds to the hairpin structure. This blocks the ribosome from binding to the mRNA, inhibiting reporter protein translation.

With increasing inducer concentration, RBP production increases, resulting in a stronger competitive inhibition of ribosomal binding to the mRNA.

Measure the different fluorescence signals from the chimeric and reporter proteins. Plot the signals, obtaining the dose-response curve.

With the inducer-mediated increase in RBP production, translational repression reduces the reporter protein concentration, confirming RNA-RBP interaction.

Transform the prepared plasmids into chemically competent bacterial cells already containing an RBP-mCerulean plasmid. To save time, plate the cells using an 8-channel pipettor on 8-lane plates containing Luria-Bertani agar with relevant antibiotics. Colonies should appear in 16 hours at 37 degrees Celsius.

Select a single colony for each double transformant and transfer to 48-well plates containing 1.5 milliliters of LB with appropriate antibiotics. Grow the cells over a period of 18 hours at 37 degrees Celsius shaking at 250 RPM. Store the overnights' glycerol stocks at -80 degrees Celsius in 96-well plates.

In the morning, program the robot to warm 180 microliters of semi-poor medium, or SPM, in 96-well plates. Program the robot to prepare the inducer plate.

In a clean 96-well plate, prepare wells with SPM consisting of 95% BA and 5% LB. The number of wells corresponds to the desired number of inducer concentrations. Add C4-HSL to the wells in the inducer plate that will contain the highest inducer concentration.

Next, program the robot to serially dilute medium from each of the highest concentration wells into 23 lower concentrations ranging from 0 to 218 nanomolar. The volume of each inducer dilution should be sufficient for all strains.

To dilute the overnight strains, program the robot to dilute by a factor of 10 by mixing 20 microliters of bacteria with 180 microliters of SPM in 48-well plates. Then, dilute again by a factor of 10 by taking 20 microliters from the diluted solution into the pre-prepared strain plate suitable for fluorescent measurements.

Now, program the robot to add the diluted inducer from the inducer plate to the 96-well plates with the diluted strains according to the final concentrations. Shake the 96-well plates at 37 degrees Celsius for 6 hours. During that time, take measurements of the optical density or OD at 595 nanometers, as well as mCherry and mCerulean fluorescence via a plate reader every 30 minutes.