In Vitro Selection of Conformational Probes for Riboswitches

Abstract

Riboswitches are non-coding RNA elements mainly located in the 5' untranslated regions (UTR) of bacterial genes. They bind to small metabolites and upon binding conformational changes occur that trigger the expression of a certain gene. Riboswitches have been identified that bind to amino acids, purines, and other small metabolites such as thiamine pyrophosphate. Riboswitches contain an aptamer domain which is necessary for interaction with the metabolite and a related expression domain which harbours structural and sequence information required for interference with gene expression. The binding of a metabolite to the aptamer domain induces structural rearrangements that are relayed to the expression domain, thereby interfering with gene expression. To investigate and determine domains of the riboswitches which undergo conformational changes upon metabolite binding we used a dynamic SELEX process and identified RNA aptamers that bind to the metabolite-free variant of the riboswitch but are released upon metabolite addition. By this means, and after determination of the binding region, domains which are necessary for proper function of a full-length riboswitch can be identified.