Dissection of conformational changes in the tetrahymena group I ribozyme and self -splicing reactions

RNA plays a central role in important biological processes, such as translation and pre-mRNA splicing. A hallmark of these processes are conformational rearrangements of the RNA. To study such changes, and to learn more about the underlying properties of RNA structure, conformational changes in the group I intron and ribozyme are dissected.;The group I ribozyme reaction is analogous to the first step of self-splicing and requires binding of 5'-splice site analog and guanosine for reversible conversion to 5'-exon analog and GA. To facilitate further dissection of the ribozyme reaction---a requirement to uncover additional conformational steps---a complete kinetic and thermodynamic framework for both reaction directions is provided.;Guanosine binding is 105 fold slower than diffusion and rate-limiting for reaction. Further analysis suggests that the guanosine binding site is not preformed but rearranges locally upon ligand binding. This provides opportunity for specificity even in the case of rate-limiting binding, as the desired substrate is shown to bind faster than the undesired substrate.;The interactions between ribozyme and substrates or products differ and provide evidence that parts of the active site are not preorganized for catalysis. The resulting reorganization is mediated through metal ion interactions and hydrogen bonds. It is suggested that hydrogen bonds and metal ions, both ubiquitous in RNAs, are well-suited for effecting and transmitting conformational changes---a possible reason that RNA conformational changes are frequent.;The contribution of a secondary structure element at the 5' and 3'-splice sites toward self-splicing is examined with a new ribozyme variant. Comparing the affinities of a series of 5'-splice site analogs to this variant suggests that the G·U pair destabilizes a secondary structure element that is dynamic in self-splicing thereby facilitating the reaction. The ability of G·U pairs to disrupt helices may hold generally for RNAs.;A self-splicing construct not limited by alternative structures is characterized and used to dissect the self-splicing reaction. Evidence for multiple conformational changes between the chemical steps is presented and possible molecular changes are discussed. It is expected that this work will provide the basis for detailed molecular analysis of conformational changes in the self-splicing reaction.