利用單分子螢光共振能量轉移技術探討不同修飾之RNA偽結與核醣體間交互作用

Abstract

偽結為一RNA二級結構，其參與許多重要的生物反應過程如RNA的複製、轉錄和轉譯等。此外，研究也發現如將偽結結構上游放置一段滑動序列，能有效刺激核醣體轉譯時發生-1框架位移，許多病毒如HIV、IBV皆是透過此方式轉譯出特定蛋白。本實驗利用由人類端粒酶RNA修改之DU177偽結做為模板，其結構上包含了兩個部分配對的莖-環，以及維持整個結構高度穩定的三重鹼基配對。在先前研究已發現當轉譯過程中，部分偽結序列進入核醣體時，會觀察到偽結結構被扭曲成一緊密的中間構形。同時，部分核醣體會因無法克服此穩定的二級結構而暫停轉譯。在本篇研究中，我們透過標定特定螢光於偽結結構上，以單分子螢光共振能量轉移技術去觀察轉譯過程中不同條件下偽結結構變化。結果發現，當去除結構中三重鹼基配對時，核醣體可輕易解開偽結，且轉譯過程中不再出現中間構形，此結果也呼應之前研究發現將三重鹼基對去除時，核醣體被誘導之-1框架位移效率為零。而在另一組實驗，我們保持偽結的三重鹼基配對但添加一段滑動序列於結構上游，在給予足夠tRNA情況下，我們預期核醣體有更多機會去解開偽結結構。結果顯示，在轉譯過程中被扭轉的中間構形同樣會出現，多數核醣體如預期皆能順利解開結構，沒有出現暫停轉譯的現象。綜合實驗結果，我們發現三重鹼基配對對於偽結來說是重要的，其能讓核醣體在轉譯過程逐步解旋時扭曲成緊密的中間構形。此外，當核醣體位於滑動序列時，會受到下游偽結影響而發生-1框架位移，幫助核醣體將偽結結構打開使轉譯不至完全停止。

Pseudoknots are a type of RNA structures that participate in many biologically important processes. They can program the ribosome to frameshift when positioned downstream of a slippery sequence. Here we use the DU177 pseudoknot, which is derived from the human telomerase RNA, as a model system. The structure of DU177 contains two partially overlapping stem-loop, and several base triples that strongly stabilize the overall structure. Our lab previously showed that during translation and ribosomal unwinding of DU177, the pseudoknot is twisted into a compact intermediate structure. The ribosome also tends to pause due to the presence of this stable “roadblock”. In my study, I performed single-molecule Förster resonance energy transfer (smFRET) to further study how base triples of DU177 induce frameshifting. I found that for the pseudoknot mutant without base triples, the ribosome unwinds the structure easily, and that the compact intermediate does not form during ribosomal unwinding. Alternatively, when a slippery sequence is inserted upstream of DU177, the intermediate structure can only form transiently as the ribosome slips and attempts to unwind DU177 multiple times on a sub-second time scale. Notably, despite the mechanical resistance of base triples, most of the ribosome molecules successfully unwind the structure after multiple attempts on the tract of the slippery sequence. In conclusion, we show that base triples serve as a key element for the formation of the compact pseudoknot intermediate that resists ribosomal unwinding and finally leads to -1 frameshifting in the presence of a slippery sequence.