2019-08-012024-05-18https://scholars.lib.ntu.edu.tw/handle/123456789/701544摘要：蛋白質的序列是由核醣體在mRNA模板上依序讀取三鹼基密碼子來決定的，但是模板上特定的序列和結構可能會刺激核醣體改變讀取軌道，進而調控兩種蛋白合成的相對量，因此一個細胞如何在蛋白合成的忠實性及可調控性之間達成平衡便顯得極為重要。為了更加了解轉譯調控的機制，我們計畫利用單分子技術去探討核醣體和核酸偽結之間的交互作用；此種偽結構造是常見的軌道移轉(frameshifting)的刺激子。首先，偽結摺疊過程中可能存在的中間體被認為是決定軌道移轉效率的決定因素，所以我們將鑑別該種結構會在何種情況下出現，以及核醣體如何影響其動態(dynamics)。其次，最近的研究發現位於核醣體上游端的結構能夠調控軌道移轉的效率，但具體過程並不清楚，因此我們將研究當一個位於下游的結構被解旋、進入核醣體、再由其上游端出現時是如何重新折疊及如何與核醣體交互作用。最後，我們將進一步確認在軌道移轉刺激子誘導下被發現的一種全新的核醣體構形。先前的實驗和分子模擬所推論的兩種新構形彼此差異頗大，我們新的實驗設計將可以區分何者較接近真實。整體而言，藉由單分子技術的特點，我們可從不同角度去探索核醣體和刺激子交互作用這個極為動態的過程，藉此更深入了解軌道移轉的分子機制與調控。<br> Abstract: The ribosome reads tri-nucleotides codons along the mRNA and catalyzes polymerization of the corresponding amino acids for protein synthesis. Maintaining the reading-frame throughout the process is the key to translational fidelity. However, specific sequences and structures on the mRNA may stimulate the ribosome to shift the reading-frame to control the relative expression levels of two proteins from a single mRNA. Thus, it is important to reach a critical balance between the fidelity and control of translation in the cell. To understand the underlying mechanism of translational regulation, we aim to use single-molecular approaches to investigate the interaction between the ribosome and the RNA pseudoknots, which are common frameshift stimulators. First, we will identify and characterize the folding/unfolding intermediates of model RNA pseudoknots, which have been implicated in determining the frameshifting efficiency, and study how the ribosome affects the structural dynamics of the intermediates and the folded pseudoknots. Second, we will probe the refolding process of simple RNA stem-loop structures when emerging from the upstream side of the ribosome during translation. Influence of upstream RNA structures to the ribosomal functions is less understood, but it has been shown to modulate frameshifting in either direction. Finally, we will explore and characterize a novel ribosomal motion induced by a frameshift stimulator. Both a previous single-molecule study and a recent computational simulation suggested that the ribosome underwent a previously unidentified motion between the subunits, but the directions proposed by these two studies were almost orthogonal. We will design a new experimental scheme that allows us to distinguish between them. Overall, here we will take the advantage of single-molecule methods to comprehensively explore the rather dynamic interactions between the ribosome and functional RNA structures, which are involved in frameshifting. The results will provide great insight into the molecular mechanisms of ribosomal reading-frame maintenance and regulation.核醣體單分子光鉗轉譯軌道移轉ribosomesingle-moleculeoptical tweezerssmFRETtranslationribosomal frameshifting