以光鉗技術研究核醣體及核醣體蛋白uS15與rpsO基因5’端未轉譯區之交互作用

Abstract

信使核醣核酸 (mRNA) 的轉譯起始作用在細胞中會受到許多不同方式來進行調控，其中一種常見的方式是藉由改變核醣核酸的結構以控制轉譯作用的進行。在本實驗中將以大腸桿菌 (Escherichia coli) 的rpsO基因以及由該基因所轉譯出的核醣體蛋白uS15作為研究目標。 uS15能夠藉由結合rpsO 信使核醣核酸的5’端未轉譯區(RPSOutr)來抑制核醣體的轉譯作用，達到調控uS15的表現量。在本篇實驗中利用光鉗技術 (Optical tweezers) 來研究uS15對於rpsO信使核醣核酸之結構動力學影響，光鉗技術能將研究目標縮小至單一分子的層級並且能夠即時觀察到兩者間的構形以及動力學變化。rpsO信使核醣核酸在一般生理環境中能夠形成雙髮夾 (double-hairpin) 或假結 (pseudoknot) 此兩種結構。在先前的研究中，RPSOutr 透過光鉗技術發現擁有四種主要的展開結構。在這裡，我們也觀察到了類似的結果。接著，我們添加了 uS15 來測試它如何影響 RPSOutr 的結構形成。實驗結果證實了uS15能夠去影響假結結構的比例以及其展開所需要的力。另外，再接續的實驗中使用了mS2L。mS2L 是 RPSOutr在第二個髮夾中具有突變，而此突變使得形成假結結構變得更加困難。然而在加入 10 μM 的 uS15 蛋白後，假結結構就得以被觀察到。 為了進一步研究RPSOutr在實際生物中的結構摺疊情形，在接續的實驗中加入了30S核醣體次單元、起始因子IF1和IF3來觀察RNA的不同摺疊變化，我們發現30S的結合可能導致髮夾結構的不穩定。此外，在30S的結合過程中，RPSOutr更難形成假結結構，導致uS15更難或無法結合。從結果中我們觀察到uS15可以穩定並促進RPSOutr假結的形成，同時30S的存在會去影響髮夾結構的形成，使的RPSOutr結構處在不穩定狀態。

Translation initiation of mRNA is regulated through different ways in the cell. Modulating the structure of mRNA is a common mechanism for cells to regulate their mRNA translation. In this study, the 5’ untranslated region (5’UTR) of the Escherichia coli (E. coli) rpsO transcript is our main research objective. E. coli ribosomal protein uS15, which is translated from the rpsO transcript, regulates its own biosynthesis by interacting with the mRNA. When the concentration of uS15 protein is in excess, the protein represses its translation through binding to the 5’UTR of rpsO mRNA (termed RPSOutr). RPSOutr can fold into either a double-hairpin or pseudoknot structure. However, ribosomal protein uS15 can only bind to the pseudoknot. To study the interaction between the mRNA and protein, here we use a single molecule technique, optical tweezers, for dynamic measurements. Optical tweezers provide us a real-time observation of a single mRNA conformational change. In previous studies, four major types of structural transitions of RPSOutr were observed via force ramping (gradually increasing the force to unfold RNA). Here we also observed similar results. Furthermore, we added uS15 to test how it affects the conformational dynamics of the RNA. The result suggests that in the presence of uS15, the proportion and unfolding force of the pseudoknot increase remarkably. mS2L, a variant of RPSOutr with mutations in the second hairpin, is also selected for further studies. Mutations on the second hairpin of the mRNA make it more difficult to form a pseudoknot structure. Our data showed that the pseudoknot structure was observed after adding 10 μM of uS15 protein. To further study the interaction of RPSOutr, 30S, initiation factors IF1 and IF3 were also added to the experiment. Different conformation changes of RNA were observed. We discovered that the binding of 30S could lead to destabilization of the upstream sequence of RPSOutr. Moreover, during the binding of 30S, RPSOutr is harder to fold into a pseudoknot structure, which caused uS15 more difficult or unable to bind the structure. In conclusion, we observed that uS15 can stabilize and promote the formation of pseudoknots of RPSOutr. Moreover, the presence of 30S affects the formation of hairpin structures, making the RPSOutr structure in an unstable state.