Investigating the structural and functional basis of catalysis in the Tetrahymena group I ribozyme

Understanding RNA catalysis requires identification and characterization of the ground state and transition state interactions that play roles in accelerating the catalyzed reaction. In this thesis, we have investigated two aspects of catalysis within the Tetrahymena group I ribozyme, the binding sites of catalytic metal ions within the ribozyme core and the specific hydrogen bonding roles played by specific 2'-hydroxyl groups.; The Tetrahymena group I ribozyme has previously been shown, using substrate atomic perturbations coupled with quantitative analysis, to employ multiple distinct metal ions to catalyze phosphoryl transfer reactions. However, the spec ligands on the ribozyme for these metal ions at the active site are not known. By performing simultaneous atomic mutations within the ribozyme's backbone and substrates followed by analysis of the observed metal rescue behavior, we have identified a specific oxygen atom, the pro -Sp phosphoryl oxygen at nucleotide C262, as a ligand for one of the three functionally identified catalytic metal ions within this ribozyme's core. Functional identification of catalytic metal ion binding sites provides insights into how RNA binds and positions metal ions for use as catalytic cofactors and, combined with recent group I intron crystal structures, opens a new window into the core architecture of this RNA metalloenzyme.; Defining the specific role(s) played by 2'-hydroxyl groups within RNA presents a formidable experimental challenge. Loss of function upon 2'-deoxynucleotide substitution establishes the importance of specific 2'-hydroxyl groups within RNA, but this approach provides no information about how these hydroxyl groups impart their functional contribution. To address the challenge of identifying 2'-hydroxyl groups that act specifically as hydrogen bond donors, we developed an atomic mutation cycle (AMC) to evaluate the functional importance of the 2'-hydroxyl group's hydrogen atom. We applied AMC analysis at the 2'-hydroxyl of the guanosine nucleophile in the Tetrahymena ribozyme reaction, and our analysis indicates this 2'-hydroxyl donates a functionally important hydrogen bond during the ribozyme reaction. The ability to define functionally important 2'-hydroxyl hydrogen bond donors within RNA offers a new strategy by which to acquire a more complete understanding of the interactions that govern RNA structure and function.