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標題: | 核醣體與訊息核醣核酸動態相嵌可促進轉譯起始過程 Dynamic Accommodation of mRNA onto Ribosomes Facilitates Translation Initiation |
作者: | Yi-Lan Chen 陳臆嵐 |
指導教授: | ?進德(Jin-Der Wen) |
關鍵字: | 轉譯起始,mRNA相嵌,轉譯起始因子,核醣體,單分子,光鉗,單分子螢光共振能量轉移, translation initiation,mRNA accommodation,initiation factor 3,ribosome,single-molecule,smFRET,optical tweezers, |
出版年 : | 2019 |
學位: | 博士 |
摘要: | 原核細胞中蛋白質的表現及恆定取決於轉譯起始的調控。轉譯起始時,核醣體會遇到擁有不同結構與序列的 mRNA。因此,了解這些不同的 mRNA 是如何與核醣體交互作用並正確地相嵌於核醣體上是一個極為重要的問題。在本研究中,透過利用單分子螢光共振能量轉移直接且即時的觀測核醣體及 mRNA 在轉譯起始時的交互作用。我們發現, mRNA 動態地來回纏繞核醣體 30S小次單元體。這個動態來回纏繞動作的頻率會因為遇到 mRNA的下游結構,像是雙股或髮夾結構,而增加。透過偵測下游結構被解開時所需要的力,我們發現,這樣的動態運動使得下游結構較不穩定。我們推測動態運動有助於核醣體有限地移動至正確的位置。透過較強的夏恩-達爾加諾序列形成的 mRNA 及核醣體交互作用可以幫助維持這樣的動態運動。
起始 tRNA的加入形成30S起始複合物 (30S IC),同時穩定 mRNA及幫助下游結構的解開至至多三個鹼基對。另外,無論起始tRNA存在與否,起始因子,尤其是 IF3 ,能幫助 mRNA 相嵌於核醣體上並抑制mRNA大幅度地運動。然而,IF3也有可能會降低 30S IC的穩定性。 在一些情況下,錯誤且穩定的 30S IC 會生成,尤其是下游出現雙股結構的時候。 這樣的雙股結構會發生在 sRNA 與 mRNA 結合時。 當這樣的情形發生時, IF3會透過移除起始轉移核醣核酸並分開mRNA及核醣體來拯救這個錯誤的複合物。在本研究中我們發現 mRNA 移動的彈性、起始密碼子及起始因子的選擇性及精確性,造就了轉譯初始發生的適切性。 Regulation of translation initiation is important for protein synthesis and protein homeostasis in prokaryotic cells. During initiation, the ribosome encounters mRNAs with different sequences and structures. Therefore, understanding how different mRNAs correctly accommodate the ribosome and maintain the fidelity of translation at the beginning of initiation is essential. In this study, by directly observing the 30S ribosomal subunit and mRNA interaction by single-molecule FRET, we found the mRNA wraps the small subunit back and forth dynamically. This movement becomes more frequent if the downstream sequence forms structures, such as duplexes and hairpins. After measuring the unfolding force of downstream structures, we found the mRNA movement during initiation helps the ribosome destabilize downstream structures. We suggest that this dynamic movement helps the ribosome to search and find correct initiation site. Interaction between mRNA and the ribosome through strong Shine-Dalgarno sequence prevents the ribosome from dropping from the mRNA during the dynamic movement. Addition of initiator tRNA forms the 30S initiation complex (30S IC). Here, initiator tRNA suppresses the dynamic movement and opens downstream structures up to 3 bp. This step is a checkpoint for the correct initiation. Initiation factors, especially IF3, also stabilizes the mRNA before and after the initiator tRNA anchoring. However, IF3 also locally destabilize the 30S IC. However, wrong stable 30S IC may form, especially when the downstream structure is a duplex structure. The duplex structures can be formed through small RNA (sRNA). In this case, IF3 can rescue the complex by removing the initiator tRNA and dissociating the mRNA-ribosome complex. Here, we reveal that the flexibility of mRNA movement, the selectivity of tRNA decoding, and the assurance of initiation factors help the ribosome maintain the robustness and fidelity of translation at the early initiation stage. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/73779 |
DOI: | 10.6342/NTU201902818 |
全文授權: | 有償授權 |
顯示於系所單位: | 基因體與系統生物學學位學程 |
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