探討核醣體30S對於能誘導框架位移之核酸偽結所產生的影響

Abstract

框架位移作用係發生於細胞內部一種轉譯重編碼現象，病毒經常會利用-1框架位移來表現特定的蛋白以及酵素(例如:SARS-CoV, HIV以及IBV)，當核醣體在進行轉譯作用時，mRNA上的特殊二級結構，例如偽結結構，可能會刺激框架位移的發生，而且框架位移的效率與偽結結構的穩定性呈現正相關。 MMTV是源自於老鼠乳腺腫瘤病毒的偽結構造，先前已經有研究團隊對它進行分析，也有利用此偽結結構進行框架位移的觀察，惟對於MMTV詳細的摺疊機制目前仍非十分清楚，且亦尚未有研究實際利用核醣體針對MMTV進行框架位移的探究，是以本實驗欲透過實際加入30S去探討對於不同框架位移效率的偽結結構，會產生何種差異性。 本研究利用單分子技術，以雷射光鉗對MMTV以及其突變體APK施加外力，並且測量解開結構所需要之外力與打開長度，進而推導其中的詳細機制，此外我們也實際加入30S以及tRNAfMet形成pre-initiation complex (pre-IC)，想去觀察其對於結構穩定性會造成何種影響。我們從實驗中發現，30S對於偽結結構的確會有實際的影響，並且在不同框架位移效率的偽結影響力也不相同，另外我們亦發現若僅加入30S反而對於結構的影響力會比pre-IC更顯著。在Constant force實驗中我們發現30S可以穩定並且幫助結構的摺疊，而且MMTV偽結結構的摺疊會分成兩步進行，此外，偽結上的第一個髮夾與結構的摺疊也有著一定的關聯性。綜合上述結果，我們推斷MMTV偽結結構的摺疊為兩步摺疊，並且是先疊第二個髮夾接著才是第一個髮夾，且在有加入30S的情況下，可以更加快速的幫助結構摺疊以及穩定。

Frameshifting is a recording translation mechanism. Viruses often use -1 frame-shifting to express specific proteins and enzymes (eg, SARS-CoV, HIV, and IBV). When the ribosome is undergoing translation, specially secondary structures on mRNA, such as pseudoknot structures, may stimulate the occurrence of frameshifting, and the efficiency of frameshifting is proportional with the stability of the pseudoknot structure. MMTV is an RNA pseudoknot derived from the mouse mammary tumor virus. It has been analyzed previously for frameshifting. However, the detailed folding mechanism of MMTV is not well understood, and no related research has been done by using ribosomes to observe the behavior of the MMTV frameshifting so far. Therefore, the main purpose of this study is dedicated on investigating the features of different frameshift-stimulating RNA pseudoknots and their responses to the action of ribosomal 30S subunit.. In this study, optical tweezers had been applied to exert external force to the MMTV and the APK. From the unfolding force and opened length, we can deduce the detailed mechanism. In addition, 30S and tRNAfMet were added to form pre-initiation complexes to observe the impact on structural stability. From the experimental outcome, 30S has prominent influence on the pseudoknot structure, and the influence can be divided into different levels according to the pseudoknot with different frameshifting efficiencies. On the other hand, we also found that if merely added 30S, the influence on the structure was more dramatic than pre-initiation complex. In experiment of Jump-drop, 30S can play a role in structure stabilization and assisting folding. Moreover, the refolding of MMTV pseudoknot occurred in two steps. Concluded by previous mention, the mechanism of refolding can be assumed by two steps. The pathway of pseudoknot formation is hp2 folded firstly, followed hp1. At the same time , the presence of 30S can accelerate the refolding and stabilize structure.