2009-12-012024-05-16https://scholars.lib.ntu.edu.tw/handle/123456789/667979摘要：位於信使核醣核酸(mRNA)上的轉譯起始區域所形成的特殊結構，通常會在基因的表現過程中扮演重要的角色。以大腸桿菌的rpsO mRNA為例，其在此區域的序列(5-UTR)會形成一個偽節(pseudoknot)或雙髮夾(double-hairpin)的結構。它的基因產物(核醣體蛋白ecS15)會結合到此mRNA所生成的偽節上並抑制其轉譯。然而就我們所知，另一個雙髮夾結構似乎沒有任何作用，除非它能經由某種調控的方式被轉變成俱功能的偽節結構。因此，研究在調控因子(包括核醣體及ecS15)存在或不存在的情況下，RNA結構如何轉換，將有助於釐清各種構形所扮演的角色。在此研究計畫中，我們將利用光箝(optical tweezers)的單分子技術來探討rpsO mRNA的動態結構，以及它和核醣體與ecS15的交互作用。此技術讓我們可以一次控制單一個RNA分子，並即時觀測其結構的改變。如此我們可以直接偵測不同的結構是否可以在同一個分子內作轉換，此特點是傳統的研究方式所難以達成的。首先我們將測量rpsO mRNA的動態特性及如何形成結構，並進而探討不同的結構是否可以互相轉換以及如何轉換。而後我們將研究核醣體及ecS15如何影響此RNA結構的形成及其穩定度。其中最有趣的是，在核醣體或ecS15存在的情況下，此RNA的雙髮夾結構是否能夠被誘導而轉換成俱生物活性的偽節結構。這些結果將有助於我們對轉譯調控的進一步了解。<br> Abstract: Structures present around the initiation region of mRNA usually play a key role in gene expression at the translation level. Regulatory factors bind to the RNA structures to activate or repress protein synthesis by the ribosome. Escherichia coli ribosomal protein S15 (ecS15, encoded by the rpsO gene), when present in excess, binds to its cognate mRNA to repress translation. The binding motif is located at the 5-untranslated region (5-UTR) of the rpsO mRNA, which folds into either a pseudoknot or a double-hairpin structure. Binding of ecS15 stabilizes the pseudoknot conformation and thus prevents the ribosome from melting it to initiate translation. In contrast, it is not known that if the ribosome or ecS15 can bind directly to the double-hairpin structure. Thus, the double-hairpin seems to play no roles in biological functions, unless it could be somehow converted to the pseudoknot by thermal fluctuation or by other factors and thus is involved in regulation. Understanding the mechanism of the RNA structural rearrangement in the presence or absence of regulatory proteins (including the ribosome and ecS15) will shed light on the molecular basis of translation regulation. In this project we propose to study the structural dynamics of the 5-UTR of the rpsO mRNA and its interaction with ecS15 as well as the ribosome using optical tweezers. This technique allows us to manipulate one single RNA molecule at a time and follow the conformational change of RNA in real time. The advantage of single-molecule experiments is to eliminate heterogeneous signals coming from the stochastic reaction nature of many molecules in bulk, thus an ideal method to study structural dynamics of macromolecules. We will first characterize the structural and dynamic aspects of the 5-UTR of the rpsO mRNA using optical tweezers and measure whether or how the pseudoknot and double-hairpin conformations are converted to each other. Next, we will investigate the interaction between the 5-UTR of the rpsO mRNA and the ribosome as well as ecS15. We will measure how the stability of RNA structures is affected upon binding by the ribosome and/or ecS15. Most interestingly, we will detect if the pseudoknot conformation could be induced to form from the double-hairpin in the presence of the ribosome or ecS15. These data will provide more insight into the nature of translation regulation with RNA structural rearrangement.核醣體轉譯光箝單分子ribosometranslationoptical tweezerssingle molecules