The self-splicing of group II introns is important for gene expression in many different organisms, from plants to bacteria. This class of RNA is of particular interest because it is autocatalytic and, as one of only seven known classes of natural ribozymes, group II introns represent a valuable resource for understanding RNA catalysis and the principles governing RNA folding. Parallels in reaction mechanism have suggested that group II introns are evolutionarily related to the eukaryotic spliceosome, and may therefore represent a simple model for complex events involved in pre-mRNA processing. Additional interest in group II introns stems from the fact that they are mobile genetic elements, capable of attacking and directly integrating themselves into genomic DNA. This reaction has important implications for the evolutionary dispersion of introns and for the potential application of group II intron ribozymes as therapeutics. Understanding of group II intron folding and active-site formation has been hindered by the fact that there are few phylogenetic covariations in Watson-Crick base- pairing that help to define the long-range interactions that stabilize intronic architecture. The most highly conserved region of the intron is a small hairpin-loop structure known as Domain 5 (D5), which contains the residues most important for catalytic activity and is widely believed to comprise the heart of the active-site. We propose a collection of experiments designed to identify the tertiary contacts that anchor D5 to the other intronic subdomains and to elucidate the structural features that contribute to chemical catalysis by D5. In previous work, we reconstructed a group II intron into a family of multiple-turnover ribozymes designed to study the chemical mechanism of catalysis. Having characterized these ribozymes, we are now poised to unravel intronic strategies for chemical reaction by specifically modifying substrates and analyzing the effects on ground- and transition-state stabilization. We propose additional experiments for understanding the hallmark reaction of group II introns: branching by a bulged adenosine. The site of adenosine docking in the core will be elucidated through a combination of biochemical mapping studies and analysis of ribozyme constructs, while the role of specific adenine substituents will be probed through single-atom changes. All proposed studies will be conducted with the ai5gamma intron and with new introns that are stable at lower ionic strength.
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