A divalent cation-dependent variant of the glmS ribozyme with stringent Ca2+ selectivity co-opts a preexisting nonspecific metal ion-binding site.

Abstract

Ribozymes use divalent cations for structural stabilization, as catalytic cofactors, or both. Because of the prominent role of Ca2+ in intracellular signaling, engineered ribozymes with stringent Ca2+ selectivity would be important in biotechnology. The wild-type glmS ribozyme (glmSWT) requires glucosamine-6-phosphate (GlcN6P) as a catalytic cofactor. Previously, a glmS ribozyme variant with three adenosine mutations (glmSAAA) 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 Ca2+-specific ribozyme (glmSCa) evolved from glmSAAA that is >10,000 times more active in Ca2+ than Mg2+, is inactive in even 100 mM Mg2+, and is not responsive to GlcN6P. This stringent selectivity, reminiscent of the protein nuclease from Staphylococcus, allows rapid and selective ribozyme inactivation using a Ca2+ chelator such as EGTA. Because glmSCa functions in physiologically relevant Ca2+ concentrations, it can form the basis for intracellular sensors that couple Ca2+ levels to RNA cleavage. Biochemical analysis of glmSCa reveals that it has co-opted for selective Ca2+ binding a nonspecific cation-binding site responsible for structural stabilization in glmSWT and glmSAAA. Fine-tuning of the selectivity of the cation site allows repurposing of this preexisting molecular feature.