Reporter Gene-Based Screening for TPP Riboswitch Activators

Methods Mol Biol. 2017;1520:227-235. doi: 10.1007/978-1-4939-6634-9_13.

Abstract

With the rise of multidrug resistant bacteria and a growing number of nosocomial infections, there has been an increased interest in finding new antibacterial drugs and drug targets. Riboswitches represent attractive new antibacterial drug targets, because they not only inherently recognize a specific metabolite or ion with their RNA aptamer domain, but also often regulate essential metabolic pathways. Here, we describe a reporter gene-based screen to identify compounds that activate the thiamine pyrophosphate (TPP) riboswitch in bacteria. This assay can be easily adapted for different riboswitch classes and thus has the potential to target many essential metabolic pathways and a broad spectrum of bacterial pathogens.

Keywords: Antibiotics; Miller assay; Thiamine pyrophosphate; Translational fusion; lacZ reporter gene.

MeSH terms

  • Cloning, Molecular
  • Enzyme Assays
  • Escherichia coli / metabolism
  • Genes, Reporter*
  • Genetic Techniques*
  • Plasmids / metabolism
  • Riboswitch*
  • Thiamine Pyrophosphate / metabolism*
  • Transformation, Genetic
  • beta-Galactosidase / metabolism

Substances

  • Riboswitch
  • beta-Galactosidase
  • Thiamine Pyrophosphate