Quantification of translation enhancement by an RNA pseudoknot structure using a lacZa reporter

Type I toxin-antitoxin (T1TA) systems are genetic modules that encode a toxic membrane protein and a small RNA antitoxin. The antitoxin inhibits toxin expression by binding to the toxin mRNA 5’UTR and preventing translation initiation. T1TAs are known to have a role in plasmid maintenance, phage defence, stress responses, and antibiotic persistence. timP/TimR is a T1TA system in which Toxic Inner Membrane Protein or TimP is a type I toxin whose expression is post-transcriptionally regulated by TimP Regulator or TimR, which is the antitoxin. TimR base pairs to a region in the 5’UTR of timP mRNA and inhibits its translation. TimR regulation is dependent on a pseudoknot structure in timP 5’UTR, which plays a dual role in both inhibition through TimR and enhancing translation initiation. In this study, we investigated the use of a lacZα reporter to quantify the effect of the pseudoknot on gene expression. A combination of molecular cloning, Western blotting and β-galactosidase assays was employed to assess and quantify the effect of the pseudoknot. Our results show that replacement of the timP ORF with lacZα entails quantification of timP gene expression. The reporter construct was regulated by TimR and sensitive to mutations abolishing pseudoknot formation. Although these findings suggest that lacZα is a valuable reporter system to study translation of type 1 toxins, the system requires further validation with respect to LacZα expression levels and reporter mRNA stability. This study provides insight into the design and quantification of a high-throughput reporter for investigating the effect of 5’UTR structural elements on gene expression.