利用單分子螢光共振能量轉移技術探討 RNA 偽結與核醣體間交互作用

Study of Interactions Between the mRNA Pseudoknot and the Ribosome with Single-Molecule FRET

謝伯思(Po-Szu Hsieh)

指導教授 ： 溫進德

國立臺灣大學/生命科學院/分子與細胞生物學研究所/碩士(2016年)

https://doi.org/10.6342/NTU201601522

摘要

偽結是一種在 RNA 中重要而複雜的結構，在各種生物反應中例如 RNA 的複製、轉錄與轉譯等等，皆扮演著重要的角色，許多 RNA 病毒更透過偽結作為刺激物，進而造成計畫性核醣體框架位移 (Programmed Ribosomal Frameshifting)。先前研究利用人類端粒酶 RNA 中所衍生的 DU177 偽結作為研究模板，其結構為兩個具有鹼基配對區域的螺旋莖 (helical stem) 透過兩個單股環 (single-stranded loop) 連接，研究發現若將此段偽結結構放置於一段滑動序列 (slippery sequence) 下游，在進行轉譯作用的過程中，可有效刺激 -1 計畫性框架位移的發生。另外， mRNA 必須呈現單股狀態才能進行轉譯作用，先前研究指出核醣體本身在轉譯作用進行時即具有解旋酶 (helicase) 的活性，可以解開 mRNA 所形成的二級結構，但目前對於偽結結構如何被核醣體解開的分子機制尚未明瞭。
本篇研究透過單分子螢光共振能量轉移技術來探討偽結結構與核醣體間的交互作用。研究結果發現核醣體在進行轉譯的過程中，當部分偽結結構序列進入核醣體時，會因受到核醣體的影響使其結構產生扭曲，這現象可能和框架位移的發生有所關聯。但隨著轉譯作用的持續進行，核醣體將會克服結構屏障進而將偽結中第一個 stem 的鹼基對全數解開。根據此結果推測，由於此偽結含有數個提高其穩定度的三重鹼基對 (base triples)，即使部分偽結的序列進入核醣體時，可能依舊保持偽結結構狀態。

關鍵字

偽結 ； 核醣體 ； 轉譯作用 ； 單分子 ； 螢光共振能量轉移技術

並列摘要

Pseudoknots are important and complex RNA structures. They play critical roles in many biological processes, such as RNA replication, transcription and translation. Several viruses use a pseudoknot structure to induce programmed ribosomal frameshifting during translation. Previous research used an H-type pseudoknot derived from the human telomerase RNA (DU177) as a model system, which has two helical stems connected by two single-stranded loops. DU177 can function as a -1 ribosomal frameshifting stimulator when it is positioned downstream of a slippery sequence. In addition, mRNA structures must be unfolded to a single-stranded form to be translated. Previous research has revealed that the ribosome has intrinsic helicase activity during translation. However, the detailed molecular mechanism of how a pseudoknot is unwound by the ribosome remains unclear.
In this study, we use single-molecule fluorescence resonance energy transfer (smFRET) to elucidate how a pseudoknot is unwound by the ribosome. Our results show that when a partial pseudoknot sequence enters the ribosome during translation, the pseudoknot structure is distorted by the ribosome. The structural distortion may play a role in inducing the ribosome to undergo frameshifting. However, the ribosome can overcome the steric barrier to unwind the first stem of the pseudoknot completely when translation proceeds further. According to our results, we propose that, due to the presence of several base triples that stabilize the whole RNA structure, the folding of the pseudoknot may be maintained even if a partial sequence enters the ribosome.

並列關鍵字

pseudoknot ； ribosome ； translation ； single-molecule ； fluorescence resonance energy transfer (FRET)

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