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|Title:||Unravelling the roles of non-Watson-Crick and triplex structures in regulating RNA folding dynamics and function||Authors:||Zhong, Zhensheng||Keywords:||DRNTU::Science::Biological sciences::Genetics||Issue Date:||2016||Source:||Zhong, Z. (2016). Unravelling the roles of non-Watson-Crick and triplex structures in regulating RNA folding dynamics and function. Doctoral thesis, Nanyang Technological University, Singapore.||Abstract:||It is not well understood how the RNA folding and unfolding dynamics, mechanical properties, and biological functions are affected by non-Watson-Crick pairs and base triples. Using a human telomerase RNA hairpin containing four consecutive non-canonical base pairs as a model system, we studied the effect of U∙U pair to U∙C pair substitutions on thermodynamic and mechanical properties. The ensemble thermal denaturation of the hairpins shows a 3-state pathway with an on-pathway intermediate with the disruption of the non-canonical base paired region as well as two apical Watson-Crick base pairs. In single molecule force spectroscopic studies, a misfolded hairpin at forces below 13 pN is observed. The single U·U to U·C substitution lowers the melting temperature of the first transition in thermal denaturation and significantly lowers the mechanical unfolding rupture force without obviously affecting the unfolding transition-state position. Furthermore, the substitution increases the frequency of misfolding and the mechanical folding rates, and shifts the folding transition-state position towards the native hairpin structure. Our results clearly demonstrate the non-nearest-neighbor effects of the U·U to U·C substitution. Many RNA viruses including Simian Retrovirus type 1 (SRV-1) utilize minus-one ribosomal frameshifting as an alternative translation mechanism to generate stable ratios of structural and catalytic proteins. The detailed mechanism of minus-one ribosomal frameshifting is still not well understood. The SRV-1 frameshifting pseudoknot is located in the overlapping region of gag and pol genes and plays critical roles in stimulating frameshifting. Using optical tweezers, we studied the mechanical properties of a series of pseudoknots derived from the wildtype SRV-1 frameshifting pseudoknot. In addition, we carried out ensemble thermal denaturation and native polyacrylamide gel electrophoresis studies. Our studies reveal that the minor-groove base triple formation in the pseudoknot enhances the structural compactness and mechanical stability. The −1 frameshifting efficiency is positively correlated with one-step unfolding rupture force and inversely correlated with the unfolding rate.||URI:||https://hdl.handle.net/10356/66532||DOI:||10.32657/10356/66532||Fulltext Permission:||open||Fulltext Availability:||With Fulltext|
|Appears in Collections:||SPMS Theses|
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