建立單分子研究之基因改造螢光核醣體

Abstract

核醣體為一個重要的胞器並存在於所有生物體內，從1953年開始被發現，後來結構陸陸續續地被解出，但是真正的轉譯作用機制尚未明瞭。為此，我們的首要目的是直接觀測核醣體與mRNA之間的交互作用，亦即如何形成複合體、轉譯時核醣體與mRNA的相互運動之結構變化，以及轉譯作用的速率等等。 我們計畫運用單分子螢光共振能量轉移技術 (smFRET)去進行上述的觀測，所以我們必須先製作一個帶有螢光分子的核醣體，在此我們以XL-10大腸桿菌株的rpsE基因做為修飾對象，將ybbR-tag利用基因置換的方式插入內生性rpsE基因3’端，使製造出的核醣體S5蛋白在C端含有ybbR-tag─可以鍵結螢光分子的平台─並透過SFP酵素將螢光分子以共價鍵方式形成穩定鍵結。另外我們也嘗試將SNAP蛋白插入核醣體S5蛋白的C端做出S5-SNAP融合蛋白以期達到在更少步驟下，取得更高螢光標定的目標。我們將修飾過的核醣體以蔗糖梯度方式純化，而mRNA的製備則從人工設計之序列胞外轉錄而來。透過螢光分析我們可以確認螢光分子鍵結在S5修飾蛋白上，利用電泳凝膠遷移分析則可以確認核醣體與mRNA正確形成複合體。冷光素酶活性分析則進一步顯示經修飾過的核醣體具有轉譯活性。最後我們得以用單分子螢光共振能量轉移技術觀測70S核醣體以起始複合體狀態結合mRNA而產生FRET訊號。我們希望藉由進一步的條件優化來改善FRET訊號比例。

Ribosomes are important machines for protein synthesis in all organisms. In the past 50 years, structures of the ribosome have been revealed by TEM, X-ray and cryo-EM. However, many aspects of the mechanism of how the ribosome translates mRNA into protein remain unclear. Here we aim to directly probe the interactions between the ribosome and mRNA by single-molecule Förster resonance energy transfer (smFRET). We choose to modify and fluorescently label the ribosomal protein S5 located at the mRNA entrance of the 30S subunit. We insert a ybbR-tag at the C terminus of the endogenous S5 protein (rpsE gene) by genomic replacement. The E. coli mutant grows normally without any apparent defects. Next, we use the SFP synthase to covalently attach Dy-547 or Dy-647 (equivalent to Cy3 or Cy5 respectively) to the ybbR-tag. Alternatively, the SNAP protein is inserted in place of the ybbR-tag, and the S5-SNAP fusion protein is overexpressed from a plasmid as an attempt to achieve better labeling efficiency with a much simpler procedure. After fluorescent labeling, we confirm the presence of the dye on the ribosome by SDS-PAGE and fluorescence imaging. The dye-labeled ribosome appears to be functional as indicated by electrophoretic mobility shift assay (EMSA) and in vitro translation with luciferase reporter assay. Single-molecule FRET signal can be observed from the initiation complex with a dye-labeled mRNA. Further optimization is needed for improvement of the labeling efficiency in order to effectively observe the interactions between the fluorescently labeled ribosome and mRNA.