Allosteric Tertiary Interactions Preorganize the c-di-GMP Riboswitch and Accelerate Ligand Binding

Cyclic diguanylate (c-di-GMP) is a bacterial second messenger important for physiologic adaptation and virulence. Class-I c-di-GMP riboswitches are phylogenetically widespread and thought to mediate pleiotropic genetic responses to the second messenger. Previous studies suggest that the RNA aptamer domain switches from an extended free state to a compact, c-di-GMP-bound conformation in which two helical stacks dock side-by-side. Single molecule fluorescence resonance energy transfer (smFRET) experiments now reveal that the free RNA exists in four distinct populations that differ in dynamics in the extended and docked conformations. In the presence of c-di-GMP and Mg²⁺, a stably docked population (>30 min) becomes predominant. smFRET mutant analysis demonstrates that tertiary interactions distal to the c-di-GMP binding site strongly modulate the RNA population structure, even in the absence of c-di-GMP. These allosteric interactions accelerate ligand recognition by preorganizing the RNA, favoring rapid c-di-GMP binding.