Ligand recognition and helical stacking formation are intimately linked in the SAM-I riboswitch regulatory mechanism

: Riboswitches are noncoding mRNA elements that control gene expression by altering their structure upon metabolite binding. Although riboswitch crystal structures provide detailed information about RNA-ligand interactions, little knowledge has been gathered to understand how riboswitches modulate gene expression. Here, we study the molecular recognition mechanism of the S-adenosylmethionine SAM-I riboswitch by characterizing the formation of a helical stacking interaction involving the ligand-binding process. We show that ligand binding is intimately linked to the formation of the helical stacking, which is dependent on the presence of three conserved purine residues that are flanked by stacked helices. We also find that these residues are important for the formation of a crucial long-range base pair formed upon SAM binding. Together, our results lend strong support to a critical role for helical stacking in the folding pathway and suggest a particularly important function in the formation of the long-range base pair.