以光鉗技術研究S15蛋白質與rpsO 基因 5’端未轉譯區核醣核酸之交互作用

Abstract

在細胞中，信使核醣核酸（mRNA）的轉譯起始作用需要受到高度調控。在此，我們聚焦於大腸桿菌（Escherichia coli）rpsO 基因所轉譯的核醣體蛋白 S15（ecS15）與其自身信使核醣核酸5’端未轉譯區之交互作用，當細胞中S15過量時此作用將導致其合成受到抑制。rpsO 基因的信使核醣核酸5’端未轉譯區可以摺疊成雙髮夾（double-hairpin）或假結（pseudoknot）兩種結構，但唯有後者可與核醣體或S15產生交互作用，而此信使核醣核酸的SD（Shine-Dalgarno）序列只會暴露於假結結構，當S15結合時，會阻擋核醣體解開此結構而抑制轉譯作用。在過去，假結結構已被廣泛研究，然而雙髮夾的作用仍了解得不夠完全。 在本篇研究，我們嘗試使用光鉗技術探究S15對rpsO 基因信使核醣核酸5’端未轉譯區之結構動力學的影響，光鉗技術使我們能夠在單分子層次即時觀察核醣核酸的構形改變。而我們發現S15的存在不只使假結結構出現的比例增加，更使解開結構所需的力大幅上升。我們推測S15能夠藉此穩定假結結構，使得核醣體無法將其解開而進行轉譯起始作用。在此實驗中，我們還嘗試重新設計光鉗實驗使用的DNA handles，使其具有大量洋地黃毒（digoxigenin）提高與珠體結合的穩定性，以因應S15結合時解開假結結構所需的較高額外施力，而我們也成功合成之，提供未來進行相關實驗時一項有用的工具。

Translation initiation of mRNA is a highly regulated process in the cell. Here, we have focused on the initiation between Escherichia coli ribosomal protein S15 (ec15, encoded by the rpsO gene) and the 5’ untranslated region (5’ UTR) of its own mRNA. The reaction results in repressing its biosynthesis when ecS15 is in excess in the cell. The 5’ UTR of rpsO mRNA can fold into two alternative structures, double-hairpin and pseudoknot, but only the latter can interact with the ribosome and S15. The Shine-Dalgarno(SD) sequence of rpsO mRNA is only exposed on the pseudoknot structure. When S15 binds to the pseudoknot, the ribosome is blocked from unfolding the structure, consequently repressing its own translation. In previous research, the pseudoknot form has been extensively studied, but the role of the double-hairpin is still not well understood. In this study, we used optical tweezers to characterize the structure dynamics of the 5’ UTR of the rpsO mRNA in the presence of S15. Optical tweezers allow us to observe RNA conformational change in real time at the single molecule level. In our results, we find that S15 not only increases the population of the pseudoknot form, but also stabilizes it such that a higher force is needed to unfold the structure. We suggest the S15 binds to the pseudoknot form to enhance the protein-RNA interaction, consequently blocking the ribosome from melting the structure to initiate translation. In addition, we also tried to design new handles used in optical tweezers experiment. The handles have many copies of digoxigenin to increase the stability betweem handles and beads. It allows us to use higher force to unfold the pseudoknot structure when S15 binds. We have successfully made the handles, which provides a useful tool in future works.