Rearrangement of a stable secondary structure during tertiary folding of the Neurospora VS ribozyme

grantor: University of TorontoThe discovery of catalytic RNAs, or ribozymes, indicated that biological catalysis is not exclusively performed by protein enzymes. Ribozymes are excellent model systems for RNA structure function analysis, since their function can be used to report on the integrity of the structure. VS RNA is an 881 nucleotide (nt) ribozyme isolated from the mitochondria of certain isolates of 'Neurospora' that performs self-cleavage and ligation reactions 'in vitro'. I developed an 'in vitro' selection system to generate functional sequence variants of a VS ribozyme for comparative sequence analysis since informative, homologous VS RNA sequences were unavailable in other isolates or other organisms. I constructed a deleted circular permutation of VS RNA that is active for ligation. RNA sequences that are active for ligation can be selectively amplified from a pool of randomized sequences. Previous observations suggested that wild-type stem-loop I undergoes a Mg2+-dependent conformational change; however, the nature of the change was unclear. To obtain information about the structure of wild-type stem-loop I required for activity, I performed 'in vitro' selection. "Active" stem-loop I sequence variants were selected from a partially-randomized pool by ligation to a VS ribozyme. A combination of comparative sequence analysis, mutational analysis, computer-based predictions, and chemical structure probing experiments were used to identify the "active" secondary structure of wild-type stem-loop I. Four of the five original base pairs in wild type stem-loop I are changed due to tertiary folding. Most RNA secondary structures are stable during tertiary or quaternary folding. This research revealed that the paradigm that RNA folding is hierarchical is not always true. Through mutational and biochemical analysis I showed that the kissing interaction in the VS ribozyme is necessary and sufficient to rearrange wild-type stem-loop I. In contrast to kissing interactions in other RNAs, which are either confined to the loops or culminate in an extended intermolecular duplex, the VS kissing interaction causes changes in intramolecular base pairs within the target stem-loop. This work revealed a new structural consequence for a common RNA tertiary motif.Ph.D