利用單分子螢光共振轉移技術探討引起框架位移之RNA偽結與核醣體間交互作用

Abstract

-1 框架位移是指核醣體在轉譯過程中重複讀取一個核苷酸，導致核苷酸密碼子重新排列組合而做出不同的蛋白質，在RNA 病毒常用來使自身的基因體有效利用的機制，發生的條件需要訊息核醣核酸 (mRNA) 的上游有滑動序列以及距離滑動序列下游5-6個核苷酸有核醣核酸的二級結構。在此實驗中我們選用∆U177偽結作為實驗材料，∆U177偽結是人類端粒酶核酸的衍生物，∆U177偽結有兩個莖、兩個環的結構以及五組三重鹼基對，在前人研究中顯示刺激-1 框架位移的效率約有52%，非常適合作為研究-1 框架位移的材料。 我們利用單分子螢光能量共振轉移技術探討在發生-1 框架位移時核醣體和下游偽結的交互作用，我們發現在核醣體解開下游偽結的過程中，偽結結構在距離核醣體P位點11-12個核苷酸時會形成扭曲的過渡態，且在核醣體中的mRNA會被拉長。若是將在第一個環和第二個莖上的三組三重鹼基對移除，偽結就不會形成扭曲的過渡態，且-1 框架位移的效率只剩4%。綜合以上結果三重鹼基對於穩定偽結結構，以及形成過度態扮演非常重要的角色，這兩個特性存在可以刺激-1 框架位移的發生。

Minus-one programmed ribosomal frameshifting (-1 PRF) occurs when the ribosome rereads a nucleotide and consequently changes the reading frame in order to properly express their genomes in many RNA viruses. To stimulate -1 PRF, the mRNA usually contains a slippery sequence with a pattern of X-XXY-YYZ and a downstream pseudoknot (PK). The ∆U177 sequence, derived from the human telomerase RNA, is folded into a pseudoknot structure with two overlapping stem-loop structures, including three major groove base triples. Previous studies have shown that the ∆U177 pseudoknot is an efficient -1 PRF stimulator. However, details about how the ribosome unwinds the downstream pseudoknot are unknown. Here, we used single-molecule Förster Resonance Energy Transfer (smFRET) to elucidate the interaction between mRNA pseudoknots and ribosomes. We found that the PK was twisted into a compact intermediate structure when the first stem of the PK was 11 to 12 nucleotides away from the P site of the ribosome and the mRNA inside the ribosome was stretched during the unwinding process. Furthermore, after disrupting the three base triples of the PK, the -1 PRF efficiency was decreased from 52% to 4% in in vitro translation experiment, the first stem of the mutant ∆U177 pseudoknot was less resistant to unwinding, and the compact intermediate structure was not observed. These results indicate that base triples would serve as a key element for the formation of intermediate structures and the maintenance of the structure stability which are important for stimulating -1 PRF. In conclusion, the flexibility of the downstream mRNA structure could greatly enhance the -1 PRF efficiency.