Browsing by Subject "Group I introns"
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Item Probing stability, specificity, and modular structure in group I intron RNAs(2010-12) Wan, Yaqi; Russell, Rick, 1969-; Browning, Karen S.; Gutell, Robin; Hoffman, David; Ren, PengyuMany functional RNAs are required to fold into specific three-dimensional structures. A fundamental property of RNA is that its secondary structure and even some tertiary contacts are highly stable, which gives rise to independent modular RNA motifs and makes RNAs prone to adopting misfolded intermediates. Consequently, in addition to stabilizing the native structure relative to the unfolded species (defined here as stability), RNAs are faced with the challenge of stabilizing the native structure relative to alternative structures (defined as structural specificity). How RNAs have evolved to overcome these challenges is incompletely understood. Self-splicing group I introns have been used to study RNA structure and folding for decades. Among them, the Tetrahymena intron was the first discovered and has been studied extensively. In this work, we found that a version of the intron that was generated by in vitro selection for enhanced stability also displayed enhanced specificity against a stable misfolded structure that is globally similar to the native state, despite the absence of selective pressure to increase the energy gap between these structures. Further dissection suggests that the increased specificity against misfolding arises from two point mutations, which strengthen a local tertiary contact network that apparently cannot form in the misfolded conformation. Our results suggest that the structural rigidity and intricate networks of contacts inherent to structured RNAs can allow them to evolve exquisite structural specificity without explicit negative selection, even against closely-related alternative structures. To explore further how RNAs gain stability from intricate architectures, we examined a novel group I intron from red algae (Bangia). Biochemical methods and computational modeling suggest that this intron possesses general motifs of group IC1 introns but also forms an atypical tertiary contact, which has been reported previously in other subgroups and helps position the reactive helix at the active site. In the Bangia intron, the partners have been swapped relative to known group I RNAs that include this contact. This result underscores the modular nature of RNA motifs and provides insight into how structured RNAs can arrange helices and contacts in multiple ways to achieve and stabilize functional structures.Item The roles of CYT-18 in folding, misfolding and structural specificity of the Tetrahymena group I ribozyme(2009-08) Chadee, Amanda Barbara; Russell, Rick, 1969-Group I introns are structured RNAs that have been used extensively as model systems for RNA folding because they are experimentally tractable, yet complex enough to have folding challenges associated with larger RNAs. The Tetrahymena group I intron consists of a set of conserved core helices and a set of peripheral elements. Peripheral elements surround the core helices and form long range tertiary contacts between each other and to the core. Interestingly, a long-lived misfolded state is populated that has the same long range tertiary contacts as the native state but differs locally within the core. Our lab showed that the intact periphery is necessary to specify the correct core structure, as mutating tertiary contacts or removing the P5abc peripheral element dramatically destabilized the native ribozyme relative to the misfolded form. However, we also showed that the thermodynamic benefit peripheral structure provided is accompanied by kinetic liability in folding, apparently because native tertiary contacts formed by peripheral elements around the misfolded core must come apart to allow refolding of the misfolded RNA to the native state. In addition to peripheral elements, proteins also play a role in stabilizing the native structures of many group I introns. The CYT-18 protein, which occupies the same binding site as P5abc, stabilizes the functional structures of certain group I introns by using a set of insertions that are absent in other related bacterial and mitochondrial aminoacyl tRNA synthetases. Using the P5abc deletion variant of the Tetrahymena ribozyme, I sought to further define CYT-18 roles in RNA folding by probing its thermodynamic and kinetic effects on the native state formation relative to the misfolded state. I demonstrated that CYT-18, like P5abc, provided thermodynamic stability to the native state. However, unlike P5abc, CYT-18 had no apparent effect on the refolding kinetics, suggesting that a protein co-factor can stabilize the functional structure without acquiring the associated costs in RNA folding kinetics. Furthermore, I found that the mechanism of CYT-18 action appears to be distinct from P5abc. Disruption of the long-range contact P14, which is formed between P5c and L2 and is part of the network of peripheral contacts, dramatically weakened P5abc binding to the native ribozyme core by ~10⁸ fold. Interestingly, CYT-18 maintained specific and tight binding to these mutants, which suggests that CYT-18 does not rely on a circular network of contacts to specifically stabilize the native state. Instead, the specificity may arise from a more direct and intimate contact of CYT-18 with the ribozyme core. This study gives insight into an evolutionary advantage of protein co-factors in RNA folding; proteins may offer thermodynamic assistance without inhibiting folding kinetics.