Application of fluorescent measurements for characterization of riboswitch-ligand interactions.

Abstract

Riboswitches are recently discovered messenger RNA motifs involved in gene regulation. They modulate gene expression at various levels, such as transcription, translation, splicing, and mRNA degradation. Because riboswitches exhibit relatively complex structures, they are able to form highly complex ligand-binding sites, which enable the specific recognition of target metabolites in a complex cellular environment. Practically in all studied cases, riboswitches use ligand-induced conformational changes to control gene expression. To monitor the structural reorganization of riboswitches, we use the local fluorescent reporter 2-aminopurine (2AP), which is a structural analog of adenine. The 2AP fluorescence is strongly quenched when the fluorophore is involved in stacking interactions with surrounding bases, and can, therefore, be used to monitor local structural rearrangements. Here, we show specific examples in which 2AP fluorescence can be used to monitor structural changes in the aptamer domain of the S-adenosyl methionine (SAM) riboswitch and where it can be used as a ligand for the guanine riboswitch.