Abstract
Riboswitches are sequences of RNA that control gene expression via RNA-ligand interactions, without the need for accessory proteins. Riboswitches consist of an aptamer that recognizes the ligand and an expression platform that couples ligand binding to a change in gene expression. Using in vitro selection, it is possible to screen large (∼ 10(13) members) libraries of RNA sequences to discover new aptamers. However, limitations in bacterial transformation efficiency make screening such large libraries for riboswitch function in intact cells impractical. Here we show that synthetic riboswitches function in an E. coli S30 extract in a manner similar to how they function in intact E. coli cells. We discovered that, although this family of riboswitches regulates the initiation of protein translation, the fate of whether an RNA message is translated is determined during transcription. Thus, ligand binding does not bias a population of rapidly equilibrating RNA structures, but rather, co-transcriptional ligand binding kinetically traps the RNA in a conformation that supports efficient translation. In addition to providing new insights into the mechanisms of action of a family of synthetic riboswitches, our experiments suggest that it may be possible to perform selections for novel synthetic riboswitches in an in vitro system.