A divalent cation-dependent variant of the glmS ribozyme with stringent Ca(2+) selectivity co-opts a preexisting nonspecific metal ion-binding site

Ribozymes use divalent cations for structural stabilization, as catalytic cofactors, or both. Because of the prominent role of Ca(2+) in intracellular signaling, engineered ribozymes with stringent Ca(2+) selectivity would be important in biotechnology. The wild-type glmS ribozyme (glmS(WT)) requires glucosamine-6-phosphate (GlcN6P) as a catalytic cofactor. Previously, a glmS ribozyme variant with three adenosine mutations (glmS(AAA)) was identified, which dispenses with GlcN6P and instead uses, with little selectivity, divalent cations as cofactors for site-specific RNA cleavage. We now report a Ca(2+)-specific ribozyme (glmS(Ca)) evolved from glmS(AAA) that is >10,000 times more active in Ca(2+) than Mg(2+), is inactive in even 100 mM Mg(2+), and is not responsive to GlcN6P. This stringent selectivity, reminiscent of the protein nuclease from Staphylococcus, allows rapid and selective ribozyme inactivation using a Ca(2+) chelator such as EGTA. Because glmS(Ca) functions in physiologically relevant Ca(2+) concentrations, it can form the basis for intracellular sensors that couple Ca(2+) levels to RNA cleavage. Biochemical analysis of glmS(Ca) reveals that it has co-opted for selective Ca(2+) binding a nonspecific cation-binding site responsible for structural stabilization in glmS(WT) and glmS(AAA). Fine-tuning of the selectivity of the cation site allows repurposing of this preexisting molecular feature.