Book Description

The past two decades of functional and structural studies on RNA enzymes have largely focused on revealing active site interactions between ribozymes and their substrates. More recently, active site interactions among ribozyme residues themselves and the substrate have revealed an intricate network of interactions in and around the active site that involves metal ions, metal ion ligands, hydrogen bonding, and stacking interactions. Does this network extend into the broader structure of the RNA? The current studies in this thesis are aimed at understanding how the overall structural scaffold of RNA is involved in catalysis. Peripheral elements that are brought into close proximity via long-range tertiary contacts surround the catalytic core of most group I introns. In the well-studied Tetrahymena group I intron, ablation of each of five long-range tertiary contacts destabilizes the folded ribozyme, indicating a role for these tertiary contacts in overall stability, as expected. But once folded, three of the five tertiary contact mutants exhibit distinct functional roles in catalysis. Structural changes distal from the mutation site, revealed by hydroxyl radical footprinting, suggest that that these contacts are coupled to the catalytic core from a distance. These structural data combined with X-ray crystal structures and phylogenetic data suggest a number of networks that structure functional sites by using long-range tertiary contacts to position rigid helices and their more local tertiary interactions relative to the rest of the RNA. Preliminary data that tests these hypotheses suggest that some, but not all, of these networks between the periphery and the functional sites may be conserved among group I introns and that long-range tertiary contacts as well as more local tertiary interactions such as base triples may act redundantly to maintain the structure of the same regions of RNA. This redundancy could allow RNAs that change conformation over their reaction cycles to rearrange only a few tertiary contacts while maintaining a global fold. With the studies described herein, we are poised to understand the structural networks that couple the peripheral regions of RNA to its catalytic sites. These insights may lead to a better understanding of how structural networks of communication are exploited to facilitate multi-step processes that are driven by more complex RNA enzymes like the ribosome and the spliceosome.