RNA is essential to many biological processes including protein biosynthesis, RNA processing, and gene expression. Its structural complexity rivals that of proteins and, in some instances, gives rise to catalytic activity. The process by which an RNA molecule folds into a functional structure and the forces that underlie RNA catalysis are still not fully understood. The objectives of this thesis were to gain a deeper understanding of the roles of metal ions and nucleobases in RNA catalysis, and the folding mechanisms of RNA in vitro and in vivo using the hepatitis delta virus (HDV) ribozyme as a model system.

Experiments were performed to probe the nature of the divalent metal ion sites in the ribozyme and any specificity for Mg²⁺. Kinetics and thermal unfolding data support the existence of two different classes of metal ion sites in the ribozyme: a structural site that is inner sphere with a major electrostatic component and a preference for Mg²⁺, and a weak catalytic site that is outer sphere with little preference for a particular divalent metal ion.

A number of experiments focused on determining the roles of nucleobases in the ribozyme. Sequence comparisons, free energy minimization, and kinetics data indicate that a base pair located at the active site does not need to be a wobble pair, as was previously suggested, and provides a structural role in catalysis. Kinetic solvent isotope effect data and proton inventories are consistent with a catalytic mechanism in which a particular cytosine residue serves as a general acid catalyst. Further efforts to understand the catalytic mechanism were made using single-molecule fluorescence resonance energy transfer (SM-FRET). A ribozyme construct was designed and prepared, and ensemble and single-molecule measurements revealed that ribozyme activity occurs under a variety of conditions. In addition, SM-FRET experiments showed that the ribozyme construct gives rise to a range of FRET efficiencies. Efforts were made to determine the molecular origins of these values. In particular, ribozyme mutants were rationally designed to influence the conformational dynamics of the system so that FRET efficiencies could be assigned to specific ribozyme structures.

The folding of the HDV ribozyme was assayed using standard in vitro assays in which full-length transcripts are renatured by heating and cooling and self-cleavage activity is initiated by addition of Mg ²⁺, and in vivo-like cotranscriptional assays in which self-cleavage activity is monitored as the RNA is synthesized. Differences in the results obtained by the two methods are discussed and a model is presented for how wild-type ribozyme sequence and flanking sequence work in concert to promote efficient self-cleavage during transcription.