Preparation and Crystallization of Riboswitches

Recent studies have revealed that the majority of biological processes are controlled by noncoding RNAs. Among many classes of noncoding RNAs, metabolite-sensing segments of mRNAs called riboswitches are unique. Discovered over a decade ago in all three kingdoms of life, riboswitches specifically and directly interact with various metabolites and regulate expression of multiple genes, often associated with metabolism and transport of small molecules. Thus, riboswitches do not depend on proteins for binding to small molecules and play a role as both metabolite sensors and effectors of gene control. Riboswitches are typically located in the untranslated regions of mRNAs where they form alternative structures in the presence and absence of the ligand and modulate expression of genes through the formation of regulatory elements. To understand the mechanism of the riboswitch-driven gene control, it is important to elucidate how riboswitches interact with cognate and discriminate against non-cognate ligands. Here we outline the methodology to synthesize riboswitch RNAs and prepare riboswitch-ligand complexes for crystallographic and biochemical studies. The chapter describes how to design, prepare, and conduct crystallization screening of riboswitch-ligand complexes. The methodology was refined on crystallographic studies of several riboswitches and can be employed for other types of RNA molecules.