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.