Natural RNA catalysts (ribozymes) perform essential reactions in biological RNA processing and protein synthesis, whereby catalysis is intrinsic to RNA structure alone or in combination with metal ion cofactors. The recently discovered glmS ribozyme is unique in that it functions as a glucosamine-6-phosphate (GlcN6P)-dependent catalyst believed to enable "riboswitch" regulation of amino-sugar biosynthesis in certain prokaryotes. However, it is unclear whether GlcN6P functions as an effector or coenzyme to promote ribozyme self-cleavage. Herein, we demonstrate that ligand is absolutely requisite for glmS ribozyme self-cleavage activity. Furthermore, catalysis both requires and is dependent upon the acid dissociation constant (pKa) of the amine functionality of GlcN6P and related compounds. The data demonstrate that ligand is integral to catalysis, consistent with a coenzyme role for GlcN6P and illustrating an expanded capacity for biological RNA catalysis.

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The glmS ribozyme resides in the 5' untranslated region of glmS mRNA and functions as a catalytic riboswitch that regulates amino sugar metabolism in certain Gram-positive bacteria. The ribozyme catalyzes self-cleavage of the mRNA and ultimately inhibits gene expression in response to binding of glucosamine-6-phosphate (GlcN6P), the metabolic product of the GlmS protein. We have used nucleotide analog interference mapping (NAIM) and suppression (NAIS) to investigate backbone and nucleobase functional groups essential for ligand-dependent ribozyme function. NAIM using GlcN6P as ligand identified requisite structural features and potential sites of ligand and/or metal ion interaction, whereas NAIS using glucosamine as ligand analog revealed those sites that orchestrate recognition of ligand phosphate. These studies demonstrate that the ligand-binding site lies in close proximity to the cleavage site in an emerging model of ribozyme structure that supports a role for ligand within the catalytic core.