Identification of functional domains and tertiary interactions in the Neurospora VS ribozyme

grantor: University of Toronto'Neurospora' VS RNA is a small catalytic RNA capable of mediating self-cleavage and ligation reactions. Like other naturally occurring small self-cleaving RNA do the products of VS cleavage bear 5' -hydroxyl and 2'-3' cyclic phosphate termini. However, VS RNA differs, from these other ribozymes in both sequence and secondary structure. The minimal contiguous sequence required for self-cleavage has been previously determined to consist of one nucleotide upstream and 153 nucleotides downstream of the canonical cleavage site. In addition to identifying secondary structure elements, structure probing of an RNA containing the minimal contiguous sequence also showed that many additional bases were protected by magnesium-dependent interactions, implying that several tertiary contacts remained to be identified. I have used site-directed mutagenesis to characterize the first long-range interaction identified in VS RNA. This interaction contains a 3 base-pair pseudoknot helix that is required for tertiary folding and self-cleavage activity of the VS ribozyme. I have further investigated the structural requirements for cis-cleavage of the VS ribozyme by designing deletions, substitutions, and circular permutations based on the secondary structure model. Four of the six helices predicted in the model have been shortened, resulting in self-cleaving RNAs of only 121-126 nucleotides. Remarkably, the shorter ribozymes exhibit a 30 to 40-fold faster 'cis'-cleavage rate. The increase in activity results from disrupting an inhibitory helix, whose 5' side contains bases upstream of the cleavage site and from constructing a circular permutation which tethers the helix containing the cleavage site to a shortened version of the rest of the ribozyme. RNAs that lack the inhibitory helix have a lower magnesium optimum than those RNAs with an intact helix, suggesting that the presence of the helix may interfere with folding, perhaps by stabilizing an inactive conformation. The non-essential regions identified by the deletions map to the periphery of a recently proposed structure model, revealing a central ribozyme core which contains the essential structural elements required for activity of the VS ribozyme.Ph.D