Biochemical Investigation of the Mechanism of Cooperative Ligand Binding by the Glycine Riboswitch: Defining the Role of RNA Aptamer Tertiary Interactions Important for Cooperativity

Cooperative ligand binding is a mechanism widely used in nature to sharpen or dampen the responsiveness of a system to changes in stimuli. Examples of cooperative binding have been extensively studied in enzymes and protein receptors but are not limited to this class of macromolecule. Riboswitches are structured RNA domains found in mRNA that are capable of binding small molecule targets effectively regulating gene expression. The glycine riboswitch forms a unique tandem aptamer structure, where each aptamer is a separate ligand-binding domain that functions cooperatively to bind glycine. We sought to understand the molecular basis of glycine riboswitch cooperativity by comparing sites of tertiary contacts in a series of cooperative and non-cooperative glycine riboswitch mutants using hydroxyl radical footprinting, in-line probing and native gel shift studies. The results illustrate the importance of a direct or indirect interaction between the P3b hairpin of aptamer 2 and the P1 helix of aptamer 1 in cooperative glycine binding. Single aptamer studies using native gel shift provide further detail on the interaction between the aptamers in conditions with and without glycine. Collectively, our data support a model in which glycine binding is sequential; where the binding of glycine to the second aptamer allows tertiary interactions to be made that facilitate binding of a second glycine molecule to the first aptamer. These results, along with the recently reported atomic resolution structures of the single glycine binding domain and tandem aptamer glycine riboswitch provide insight into cooperative ligand binding in a novel RNA system.