核醣體轉譯框架位移與下游阻礙因子關係之研究

Steric Hindrance-Induced Ribosomal Frameshifting

王駿(Jyun Wang)

指導教授 ： 温進德

國立臺灣大學/生命科學院/分子與細胞生物學研究所/碩士(2018年)

https://doi.org/10.6342/NTU201803058

摘要

在mRNA進行轉譯的時候，會以三個鹼基為一組密碼子進行轉譯，當核醣體碰撞到下游的二級結構，就有機會在滑動序列的位置產生框架位移。框架位移會使得核醣體讀取鹼基的方式與原先不同，並且生成不同的蛋白產物。
關於框架位移的研究大多在探討甚麼因子能引起框架位移，或是會造成甚麼影響。對於產生框架位移的機制仍不了解，但我們依據二級結構與蛋白引起的框架位移現象推測與轉譯時碰撞到下游的障礙有關，因此我們以鍵結RNA的蛋白作為障礙物，並且設計能讓蛋白鍵結的序列，觀察轉譯時框架位移發生的狀況。
一開始我們讓帶有Nsp1β鍵結序列 (C-rich) 的mRNA與Nsp1β蛋白共同進行轉譯實驗。我們認為在Nsp1β蛋白結合到C-rich上後能作為障礙，核醣體轉譯時碰撞到Nsp1β蛋白時會促使-1框架位移發生。但在我們操作一連串的細胞內、外轉譯實驗後，並沒有看見-1框架位移效率有顯著的提升。
於是我們將目標轉向帶有Nsp1β鍵結序列的mRNA，發現在細胞內轉譯的狀況下滑動序列與C-rich距離較短者，其-1框架位移的效率較原本長度高。但令人驚訝的是在細胞外轉譯中卻沒有太大差別，於是我們懷疑有其他蛋白在參與框架位移的發生。我們發現核醣體蛋白S1具有鍵結C-rich序列的傾向，且在細胞外轉譯中加入S1蛋白使得-1框架位移的比例上升。不過沒辦法很好地再現實驗。我們認為轉譯試劑中原先存在的S1蛋白會影響實驗結果，在嘗試減少試劑中的S1蛋白後，發現-1框架位移的效率下降，就上述的實驗，我們認為S1蛋白能以路障的方式促使框架位移提升。為了提出更有說服力的結論，我們嘗試純化去除S1蛋白的核醣體，並用此核醣體來進行細胞外轉譯實驗。若沒有S1蛋白在轉譯時，框架位移效率會有明顯下降，重新加入S1蛋白後又能回復框架位移的效率。
在細菌、病毒甚至人體中都有用框架位移來調控轉譯的現象，或許清楚機制後，能用於消滅病原或是調控人體基因的表現。

關鍵字

核醣體 ； 框架位移 ； 轉譯 ； Nsp1β ； 核醣體蛋白S1

並列摘要

During translation, programmed ribosomal frameshifting occurs when the ribosome encounters a secondary structure downstream from a slippery sequence. Despite decades of research, the mechanistic details on how frameshifting is induced remain unclear. Here we ask whether frameshifting can be induced with a non-RNA obstacle. We use the RNA-binding protein and an mRNA with the protein target site (the C-rich motif) downstream from a slippery sequence as our model system.
First, we performed in vitro translation of the target sequence in the presence of the purified Nsp1β. Next, we assembled the genes of nsp1β and the target sequence into a single plasmid by PCR, and shortened the distance between the slippery sequence and C-rich motif to express the protein together with the target mRNA. In these experiments, we did not see the -1 frameshifting efficiency strongly enhanced. Then we turned our attention to the construct with the C-rich motif. We found that shortening the distance between slippery sequence and C-rich motif increases -1 frameshifting efficiency in vivo. Surprisingly, there was no difference in vitro. We suspected there were other proteins that could induce frameshifting. We found that ribosomal protein S1 has the tendency of binding to C-rich sequences. When we added S1 protein during translation, the -1 frameshifting efficiency was enhanced. We think the S1 protein in the translation kit may influence the results. When we sequestered the S1 protein from the kit, the frameshifting efficiency was dropped. To further confirm this result, we purified the ribosomal protein S1 depleted ribosome and used the purified ribosome to perform in vitro translation experiments. The preliminary data show that the translation activity was low and thus the frameshifting efficiency was difficult to assess.
According to our results, we reason that ribosomal protein S1 binds to the C-rich sequence and act as a road block that may induce -1 ribosomal frameshifting.

並列關鍵字

ribosome ； frameshift ； translation ； Nsp1β ； ribosomal protein S1

參考文獻

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