Characterization of metabolite-sensing riboswitches using in vivo reporter gene assays.

Riboswitches are genetic regulatory elements based on RNA molecules that control the expression of genes. Most known metabolite-sensing riboswitches control expression of genes involved in anabolic reactions but a few also control catabolic reactions. In addition, riboswitches control the expression of genes encoding metabolite uptake systems. Binding of a specific small molecule ligand (<1500 Da) to the aptamer ("binder") part of the riboswitch typically results in repression of the downstream genes, in many cases by either transcriptional termination or inhibition of translation. The downstream expression control is supported by the so called "expression platform" which is part of the riboswitch. In this chapter the analysis of thiamine pyrophosphate (TPP)-responsive riboswitches using a specialized dual-luciferase reporter gene assay is exemplarily described and may serve as a blueprint to study other bacterial riboswitches. Also, the mode of action of the riboswitches can be determined by testing transcriptional as well as translational gene fusions using different test plasmids. The effect of candidate ligands which negatively interfere with riboswitch functions can be investigated using this assay, provided the chemicals are taken up by the test organism. The assay can be thus to identify and characterize novel compounds that have the capacity to act as antibiotics. As riboswitches constitute widespread genetic control elements, they represent novel targets for antimicrobial compounds.