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 (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 Ca²⁺-specific ribozyme (glmS^Ca) evolved from glmS^AAA 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 glmS^Ca functions in physiologically relevant Ca²⁺ concentrations, it can form the basis for intracellular sensors that couple Ca²⁺ levels to RNA cleavage. Biochemical analysis of glmS^Ca reveals that it has co-opted for selective Ca²⁺ 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.

Keywords: SAXS; catalytic RNA; in vitro selection; molecular exaptation; phosphorothioate interference.