Chemical modification structure probing and interference assays for structural and functional studies of the Neurospora VS ribozyme

grantor: University of TorontoNeurospora VS RNA is a naturally occurring catalytic RNA molecule, which is capable of both RNA-mediated cleavage and ligation reactions. Like the hammerhead, hairpin and hepatitis delta ribozymes, VS RNA cleaves to generate $2\sp\prime3\sp\prime$-cyclic phosphate and 5$\sp\prime$-hydroxyl termini; however VS RNA is not similar in sequence to these other self-cleaving motifs. Chemical modification structure probing coupled with previous mutational data was used to construct a model for the secondary structure of the self-cleaving region of VS RNA is proposed which is distinct from the other small self-cleaving RNA motifs. I have also identified several magnesium-dependent interactions that form during the folding of VS RNA into its active structure. The identification of the nucleotides that were protected from chemical modification in the presence of magnesium suggested that a number of tertiary interactions were being formed; however, the identity of these interactions had not yet been elucidated. Using chemical modification of the wild type and certain VS RNA mutants I have helped to identify a three base-pair pseudoknot helix that is required for the self-cleavage and tertiary folding of VS RNA. This is the first identified long-range tertiary interaction in VS RNA. In order to identify functionally important nucleotides and structural elements of VS RNA I used a base-specific damage selection (modification interference) assay. Many modified bases that interfered with activity were clustered at helix junctions, certain internal loops and in the long-range pseudoknot; these correspond to previously determined sites of magnesium-dependent protections. This clustering indicates that the bases are required not only because they contribute to a large number of individual interactions, but because they form a smaller number of structural elements that are important for activity. I also found a separate cluster of bases whose modification enhanced cleavage. These bases appear to form a structural element that naturally attenuates the cleavage reaction. Structure probing studies indicate that stem-loop I (which contains the cleavage site) forms only in the absence of magnesium and that an alternative structure forms in the presence of magnesium. This alternative structure involves at least a partial unwinding of stem-loop I that allows for the formation of the pseudoknot. I have examined the folding of VS RNA under a variety of different magnesium concentrations and in different constructs to examine the magnesium-dependent conformational changes that accompany the formation of the catalytically active structure of VS RNA. I have also demonstrated directly by constructing a number of multiple substitution-mutations that disrupt stem I that base pairing in stem I is not essential for self-cleavage; most of these mutants have only a minimal effect on the rate of self-cleavage.Ph.D