Exploring how the ribosomal 30S subunit affects two frameshift-stimulating pseudoknot RNA
| Autor: | Tsung-Tse Wu, 吳宗澤 |
|---|---|
| Rok vydání: | 2018 |
| Druh dokumentu: | 學位論文 ; thesis |
| Popis: | 106 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. |
| Databáze: | Networked Digital Library of Theses & Dissertations |
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