title: Measuring riboswitch activity in vitro and in artificial cells with purified transcription-translation machinery.
abstract: We present a simple method to measure the real-time activity of riboswitches with purified components in vitro and inside of artificial cells. Typically, riboswitch activity is measured in vivo by exploiting β-galactosidase encoding constructs with a putative riboswitch sequence in the untranslated region. Additional in vitro characterization often makes use of in-line probing to explore conformational changes induced by ligand binding to the mRNA or analyses of transcript lengths in the presence and absence of ligand. However, riboswitches ultimately control protein levels and often times require accessory factors. Therefore, an in vitro system capable of monitoring protein production with fully defined components that can be supplemented with accessory factors would greatly aid riboswitch studies. Herein we present a system that is amenable to such analyses. Further, since the described system can be easily reconstituted within compartments to build artificial, cellular mimics with sensing capability, protocols are provided for building sense-response systems within water-in-oil emulsion compartments and lipid vesicles. Only standard laboratory equipment and commercially available material are exploited for the described assays, including DNA, purified transcription-translation machinery, i.e., the PURE system, and a spectrofluorometer.
authors.affiliation: CIBIO, University of Trento, Mattarello, Italy.
authors.forename: Laura
authors.initials: L
authors.lastname: Martini
authors.forename: Sheref S
authors.initials: SS
authors.lastname: Mansy
mesh_terms: D058045:Artificial Cells / Q000737:chemistry / Q000166:cytology / Q000378:metabolism*; D001483:Base Sequence; D008081:Liposomes / Q000737:chemistry / Q000378:metabolism; D008856:Microscopy, Fluorescence / Q000379:methods; D008969:Molecular Sequence Data; D014176:Protein Biosynthesis; D058928:Riboswitch; D014158:Transcription, Genetic
chemical_list: D008081:Liposomes; D058928:Riboswitch