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標題: | 以單分子技術與理論計算研究由信使核糖核酸結構引導之轉譯再編碼 Single-Molecule and Theoretical Approaches Reveal Translational Recoding Guided by mRNA Dynamics |
作者: | Kai-Chun Chang 張凱鈞 |
指導教授: | 溫進德(Jin-Der Wen) |
關鍵字: | 核醣體,單分子技術,框架位移,異位調控,RNA結構,分子動態模擬,計算生物學, ribosome,pseudoknot,frameshifting,single-molecule FRET,MD simulations,allostery,RNA folding, |
出版年 : | 2018 |
學位: | 博士 |
摘要: | 在許多病毒、細菌甚至人體細胞中,核醣體轉譯框架位移可以被穩定的mRNA偽結之特殊結構有效地誘導。框架位移不僅可被生物體用來調節mRNA穩定性,更可以從一條mRNA上轉譯出多條不同功能的蛋白質。然而究竟mRNA偽結的何種性質-例如熱力學穩定度、反摺疊機械力、亦或是結構可塑性-會決定框架位移效率仍有許多爭議。我們使用人類端粒酶RNA的偽結(框架位移效率約50%)作為研究模型,並使用單分子螢光共振能量轉移以及計算建模與模擬來研究偽結的摺疊和反摺疊。
我們首先架設了全內反射螢光顯微鏡作為單分子螢光共振能量轉移的實驗平台,並利用此系統發現了偽結一端的三鹼基配對可以穩定該分子內另一端的三鹼基配對,並降低其結構機動性。此分子內的協調交互作用可能是先前在單分子光鉗上觀察到偽結單步驟反摺疊的主因。有趣的是,當偽結在沒有其他外力下被核醣體逐步解旋時,會有一較緊密的中間構型形成。藉由計算模擬技術,我們發現此中間構型的形成是由於該偽結會被核醣體蛋白質次單元S3上帶正電的胺基酸吸引,然而當三鹼基配對被突變破壞掉之後,此吸引作用力就會消失,中間構型也跟著無法形成,框架轉移效率更從突變前的50%下降到0%。 利用計算建模,我們進一步發現框架轉移是由於偽結推動核醣體30S小次單元進行滾動所造成,而三鹼基配對突變的偽結既無法形成中間構型,也無法有效推動30S小次單元的滾動動作。如此從原子到分子的層面我們都圓滿地解釋了偽結如何能促進框架位移,而何以突變偽結不行。 最後,除了框架位移,核醣體更可滑動50個鹼基進行核醣體轉譯略過,而不轉譯其中對應的胺基酸序列。在此我也用了如以上所述的實驗技術來探討轉譯略過的過程。我發現在此時核醣體的解旋活性會完全消失以進行滑動,而在降落密碼子下游的終止密碼子存在與否也可能影響轉譯略過的效率。 In many viruses and bacteria, stable mRNA pseudoknots (PKs) are known to induce ribosomal frameshifting (FS) for synthesizing different proteins from the same mRNA. However, what structural features (thermal stability, mechanical unfolding force and/or structural plasticity) of a pseudoknot play dominant roles in determining FS efficiency remains controversial. Here we use an FS-inducing pseudoknot derived from the human telomerase RNA (hTR-PK) as our model system. The unfolding and folding dynamics of hTR-PK and its mutant derivatives are investigated with single-molecule Förster resonance energy transfer (smFRET) and computational methods. For smFRET, we set up a total internal reflection fluorescence microscope and used it to show that base triples at one end of hTR-PK promote the formation of those at the other, and thereby limit the flexibility of the latter. The coordination between base triples is therefore responsible for the single-step unfolding event of the structure seen in optical tweezers experiments. By contrast, smFRET experiments identify a compact intermediate structure before hTR-PK is completely disrupted by the ribosome. Steered molecular dynamics simulations (SMD) further reveal that as the loop of hTR-PK attaches to the positively charged residues of ribosomal protein S3, base triples facilitate the formation of the hTR-PK unfolding intermediate. When the base triples are disrupted by mutations (the delta triple mutant), the compact unfolding intermediate can no longer be seen in SMD simulations and smFRET experiments (that is, the mutant exhibits single-step unfolding). The delta triple mutation also results in a dramatic drop in FS efficiency from ~50% to ~0%. Our study demonstrates the importance of a base triple-stabilized unfolding intermediate in PK-induced FS. Through computational modeling, we further discover that PK-induced 30S subunit rolling is responsible for FS. When the base triples are disrupted, the unfolding intermediate does not form, and therefore can no longer induce subunit rolling, which in turn leads to the drop in FS efficiency. Our results provide mechanistic insights on how PK induces conformational change of the ribosome at single-residue level during FS. Finally, in addition to FS, the ribosome can also bypass a specialized 50-nt-long mRNA sequence. Here I applied similar experimental techniques described above to investigate how bypassing occurs. I found that the ribosomal helicase activity is completely disrupted after take-off and that, unexpectedly, the presence of the stop codon downstream of the landing codon can also affect bypassing efficiency. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/20060 |
DOI: | 10.6342/NTU201801101 |
全文授權: | 未授權 |
顯示於系所單位: | 分子與細胞生物學研究所 |
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