2017-08-012024-05-18https://scholars.lib.ntu.edu.tw/handle/123456789/706376摘要：轉譯起始階段的調控是細胞維持其內部蛋白濃度所必需的，因其重要性，該反應的整個過程在過去已有相當程度的研究。然而仍有許多極為基本的性質尚待進一步釐清，例如核醣體的小次單元30S如何在mRNA模板上尋找包含SD序列和起始密碼子的結合位至今仍不清楚。在這裡我們將使用單分子技術研究起始階段分子間的交互作用，包含mRNA、30S、起始tRNA、核醣蛋白S1、以及一些起始因子(特別是IF3)。我們會將一對螢光染劑同時標定在mRNA的兩端，或mRNA和30S各一，接著利用smFRET測量在30S或其他因子結合在mRNA之後的構形變化，另外我們也將使用光鉗觀察RNA結構在30S作用下的動態。藉著此種方法學上的特色，我們預期將可分辨30S在最初的搜尋階段是會沿著mRNA在上面滑行？或是跳上跳下直到遇見結合位？S1蛋白在此過程中所扮演的角色也可進一步確認。此外，我們將測量mRNA如何藉著起始tRNA的結合而纏繞在30S上形成穩定的前初始複合體(pre-initiation complex)。最後，我們將針對大腸桿菌中的一個模式轉譯調控系統，探究其有效率的轉譯反應是如何達成的。整體而言，這些研究結果將會讓我們對細菌內轉譯起始的分子機制有更深一層的了解。<br> Abstract: The initiation stage is a major point where a translation reaction is regulated. Such a regulation is required to well maintain the protein level in the cell. Due to its importance, the whole process has been extensively studied. However, some fundamental features remain elusive. For example, how the small subunit 30S of the ribosome search the mRNA template for the binding site, consisting of the Shine-Dalgarno sequence and start codon, is not clear. In this project, we will mainly use single-molecule approaches to explore the interactions among the mRNA, 30S subunit, initiator tRNA, ribosomal protein S1, and some of the initiation factors (especially IF3) in the beginning of the translation initiation. We will label a pair of fluorescent dyes on the mRNA or between the mRNA and 30S to measure conformational change after binding of the 30S or other factors using single-molecule FRET (smFRET). Optical tweezers will also be used to measure structural changes of the mRNA under the action of the 30S. Taking advantages of the methodology, we will be able to distinguish if the 30S subunit slides along or hops on-and-off from the mRNA during the searching process. At which point the S1 protein plays a role in this process will also be clarified. In addition, we will measure wrapping of the mRNA on the 30S when the initiator tRNA is present; this is to form the stable pre-initiation complex. Finally, we will use a model system of the initiation regulation in E. coli to investigate how effective translation can be achieved. Our results will provide great insight into the molecular mechanisms of translation initiation in bacteria.核醣體單分子光鉗轉譯ribosomesingle-moleculeoptical tweezerssmFRETtranslation