title: Fluorescence monitoring of riboswitch transcription regulation using a dual molecular beacon assay.
abstract: Riboswitches are mRNA elements that specifically bind cellular metabolites and control gene expression by modifying their structure. As riboswitches often control essential genes in pathogenic bacteria, riboswitches have been proposed as new targets for antibiotics. High-throughput screening provides a powerful approach to identify riboswitch ligand analogs that could act as powerful antibacterial drugs. Biochemical assays have already been used to find riboswitch-binding analogs, but those methods do take into account the transcriptional context for riboswitch regulation. As the importance of co-transcriptional ligand binding has been shown for several riboswitches, it is vital to develop an assay that screens riboswitch-binding analogs during the transcriptional process. Here, we describe the development of a dual molecular beacon system monitoring the transcriptional regulation activity of the Bacillus subtilis pbuE adenine riboswitch. This system relies on two molecular beacons that enable the monitoring of transcription efficiency, as well as the regulatory activity of the riboswitch. Different analogs were tested using our system, and a good correlation was observed between riboswitch activity and reported metabolite affinities. This method is specific, reliable and could be applied at the high-throughput level for the identification of new potential antibiotics targeting any riboswitch-regulating gene expression at the mRNA level.
authors.affiliation: Department of Biology, Faculty of Sciences, RNA Group, Université de Sherbrooke, Sherbrooke, Quebec J1K 2R1, Canada.
authors.forename: Raja
authors.initials: R
authors.lastname: Chinnappan
authors.forename: Audrey
authors.initials: A
authors.lastname: Dubé
authors.forename: Jean-François
authors.initials: JF
authors.lastname: Lemay
authors.forename: Daniel A
authors.initials: DA
authors.lastname: Lafontaine
mesh_terms: D000225:Adenine / Q000031:analogs & derivatives / Q000494:pharmacology; D001412:Bacillus subtilis / Q000235:genetics; D004353:Drug Evaluation, Preclinical; D005456:Fluorescent Dyes / Q000737:chemistry*; D005786:Gene Expression Regulation / Q000187:drug effects*; D006147:Guanine / Q000031:analogs & derivatives / Q000494:pharmacology; D008024:Ligands; D015341:Nucleic Acid Probes / Q000737:chemistry*; D033702:Nucleobase Transport Proteins / Q000235:genetics; D058928:Riboswitch / Q000187:drug effects*; D013050:Spectrometry, Fluorescence; D014158:Transcription, Genetic / Q000187:drug effects*
chemical_list: D005456:Fluorescent Dyes; D008024:Ligands; D015341:Nucleic Acid Probes; D033702:Nucleobase Transport Proteins; D058928:Riboswitch; D006147:Guanine; D000225:Adenine