- 學位論文
以單分子螢光共振能量轉移技術研究核醣體在轉譯起始階段的搜尋機制
The study of the 30S subunit searching mechanism in translation initiation by single-molecule FRET
摘要
轉譯作用的起始階段在於調控蛋白質生成中扮演著舉足輕重的地位，作為一個速率決定步驟，它掌控了轉譯作用的效率以及準確性。在轉譯作用的起始階段中，許多信使RNA都使用一段夏因-達爾加諾序列使得核醣體得以辨識。夏因-達爾加諾序列通常坐落於起始密碼子 (AUG) 上游六個核苷酸的位置，並得以透過與16S 核醣體RNA上的夏因-達爾加諾序列互補序列互相鍵結進而穩定30S 核醣體次單元於信使RNA上。在30S次單元坐落於信使RNA後，便可於附近搜索適當的位置使得起始密碼子得以適當地位於核醣體P位點中。先前的研究已經了解轉譯起始因子(IF1, IF2 and IF3) 以及起始tRNA是如何參與在轉譯起始階段，然而信使RNA如何去徵調30S次單元以及30S次單元是如何於信使RNA上搜尋並到達夏因-達爾加諾序列的機制仍鮮為人知。在此，我們利用單分子螢光共振能量轉移技術進行研究，並展現了夏因-達爾加諾序列本身並不足以徵調核醣體。結合即時訊號的觀察，我們提出一個假說：30S次單元會先透過非專一性結合的方式坐落於轉譯起始位置兩側的序列上，進而移動至轉譯起始位置並完成轉譯起始階段。
並列摘要
Translation initiation is a key step for regulating protein synthesis. As a rate limiting step, it controls not only translation efficiency but also fidelity. During translation initiation, many mRNAs employ a purine-rich Shine-Dalgarno (SD) sequence, which is usually located 6 nucleotides upstream of the start codon (AUG), to base pair with the anti-Shine-Dalgarno sequence (aSD) on 16S rRNA of the ribosomal 30S subunit. This SD-aSD interaction allows 30S subunits to bind to the mRNA stably and search locally for an appropriate start codon to locate in the P site of the 30S subunit. Many previous studies have shown how translation initiation factors (IF1, IF2 and IF3) and initiator tRNA participate in translation initiation. However, little is known about how the mRNA recruits 30S subunits and how 30S subunits move along the mRNA to the SD sequence and start site. Here, by using single molecule Förster Resonance Energy Transfer (smFRET), we demonstrated that the SD sequence alone was not sufficient to recruit ribosomes and either a long, single-stranded sequence or long, structured sequences could help recruit ribosomes. Combining real-time observations, we proposed that the 30S must first bind to flanking sequences nonspecifically and travel to the initiation start site to accomplish initiation.