Structure-function relationships in RNA: Bacterial group I intron folding and active site protonation in a genomic HDV ribozyme

Hepatitis delta virus ribozymes have been proposed to perform self-cleavage via a general acid base mechanism involving an active-site cytosine, based on evidence from both a crystal structure of the cleavage product and kinetic measurements. To determine whether this cytosine (C75) in the genomic ribozyme has an altered pK_a consistent with its role as a general acid or base, 13C NMR was used to measure its microscopic pK _a in four forms of the ribozyme. The measured pK_a is moderately shifted from that of a free nucleoside or a base-paired cytosine, and has the same divalent metal ion dependence as the apparent reaction pK_as measured kinetically. However, under all conditions tested, the microscopic pK_a is lower than the apparent reaction pK_a, supporting a model in which C75 is deprotonated in the product form of the ribozyme at physiological pH. Additional results suggest that the pK_a is not shifted in the reactant state of the ribozyme and further suggest that elevated C75 pK_a may be associated with an intermediate state of the transesterification reaction.; Magnesium is required for both folding and catalytic activity of group I introns. Magnesium ion binding sites in the small group I introns from Azoarcus (205 nt) and Anabaena (249 nt) are studied with the goal of identifying those ions involved strictly in RNA structure versus those required for catalysis. Using assays designed to assess intron folding and catalytic activity independently, phosphorothioate interference mapping is used to identify phosphate oxygens that may coordinate functionally important magnesium ions. Comparison of the sites of interference observed in each assay shows (1) that the cores of the two bacterial introns have the similar but different chemical phylogeny and (2) indicates sites that interfere exclusively with global folding or catalysis and sites that interfere with both folding and catalysis.; Solids-state nuclear magnetic resonance is introduced as a novel tool to study stereochemistry of ribonucleic acids. Preliminary experiments yielding one-dimensional spectra of an RNA duplex derived from the HIV trans-activating response region and two-dimensional 1H-13C heteronuclear correlation spectra of cytosine nucleoside suggest that full assignment of individual chemical sites in a moderately-sized RNA molecule may be achieved in the near future.