title: A divalent cation-dependent variant of theglmSribozyme 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-typeglmSribozyme (glmSWT) requires glucosamine-6-phosphate (GlcN6P) as a catalytic cofactor. Previously, aglmSribozyme 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 fromglmSAAAthat 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 fromStaphylococcus, allows rapid and selective ribozyme inactivation using a Ca2+chelator such as EGTA. BecauseglmSCafunctions in physiologically relevant Ca2+concentrations, it can form the basis for intracellular sensors that couple Ca2+levels to RNA cleavage. Biochemical analysis ofglmSCareveals that it has co-opted for selective Ca2+binding a nonspecific cation-binding site responsible for structural stabilization inglmSWTandglmSAAAFine-tuning of the selectivity of the cation site allows repurposing of this preexisting molecular feature.
authors.affiliation: National Heart, Lung and Blood Institute, Bethesda, Maryland 20892-8012, USA.
authors.forename: Matthew W L
authors.initials: MW
authors.lastname: Lau
authors.affiliation: National Heart, Lung and Blood Institute, Bethesda, Maryland 20892-8012, USA.
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Trachman
authors.affiliation: National Heart, Lung and Blood Institute, Bethesda, Maryland 20892-8012, USA.
authors.forename: Adrian R
authors.initials: AR
authors.lastname: Ferré-D'Amaré
mesh_terms: D020029:Base Pairing; D001483:Base Sequence; D001665:Binding Sites; D055162:Biocatalysis; D002118:Calcium / Q000737:chemistry*; D065096:Calcium Chelating Agents / Q000737:chemistry; D002413:Cations, Divalent; D003067:Coenzymes / Q000737:chemistry*; D004492:Edetic Acid / Q000737:chemistry; D005944:Glucosamine / Q000031:analogs & derivatives / Q000737:chemistry; D019298:Glucose-6-Phosphate / Q000031:analogs & derivatives / Q000737:chemistry; D007700:Kinetics; D008274:Magnesium / Q000737:chemistry; D009690:Nucleic Acid Conformation; D054735:Phosphorothioate Oligonucleotides / Q000737:chemistry; D011485:Protein Binding; D012313:RNA / Q000737:chemistry*; D059367:RNA Cleavage; D016337:RNA, Catalytic / Q000737:chemistry* / Q000648:ultrastructure; D016415:Sequence Alignment
chemical_list: D065096:Calcium Chelating Agents; D002413:Cations, Divalent; D003067:Coenzymes; D054735:Phosphorothioate Oligonucleotides; D016337:RNA, Catalytic; C001293:glucosamine 6-phosphate; D019298:Glucose-6-Phosphate; D012313:RNA; D004492:Edetic Acid; D008274:Magnesium; D005944:Glucosamine; D002118:Calcium
keywords: SAXS; catalytic RNA; in vitro selection; molecular exaptation; phosphorothioate interference