Study of Interactions Between Different Modifications of mRNA Pseudoknots and the Ribosome by Single-Molecule FRET

• Main Authors: An-I Lee, 李安益
• Other Authors: Jin-Der Wen
• Format: Others
• Language: zh-TW
• Published: 2018
• Online Access: http://ndltd.ncl.edu.tw/handle/6388ky

碩士 === 國立臺灣大學 === 分子與細胞生物學研究所 === 106 === Pseudoknots are a type of RNA structures that participate in many biologically important processes. They can program the ribosome to frameshift when positioned downstream of a slippery sequence. Here we use the DU177 pseudoknot, which is derived from the human telomerase RNA, as a model system. The structure of DU177 contains two partially overlapping stem-loop, and several base triples that strongly stabilize the overall structure. Our lab previously showed that during translation and ribosomal unwinding of DU177, the pseudoknot is twisted into a compact intermediate structure. The ribosome also tends to pause due to the presence of this stable “roadblock”. In my study, I performed single-molecule Förster resonance energy transfer (smFRET) to further study how base triples of DU177 induce frameshifting. I found that for the pseudoknot mutant without base triples, the ribosome unwinds the structure easily, and that the compact intermediate does not form during ribosomal unwinding. Alternatively, when a slippery sequence is inserted upstream of DU177, the intermediate structure can only form transiently as the ribosome slips and attempts to unwind DU177 multiple times on a sub-second time scale. Notably, despite the mechanical resistance of base triples, most of the ribosome molecules successfully unwind the structure after multiple attempts on the tract of the slippery sequence. In conclusion, we show that base triples serve as a key element for the formation of the compact pseudoknot intermediate that resists ribosomal unwinding and finally leads to -1 frameshifting in the presence of a slippery sequence.