單分子螢光共振能量轉移應用於rpsO基因轉錄本形成轉譯前起始複合物之動態分析

Abstract

大腸桿菌中編碼核醣體S15蛋白的rpsO基因，其轉錄本的5’端未轉譯區能形成雙髮夾 (double-hairpin) 及偽結 (pseudoknot) 兩種二級結構來調控其自身的轉譯作用，過去研究認為只當偽結形成時核醣體小次單元30S才能與之結合以進行後續的轉譯，但當S15蛋白表現過量時，其會與rpsO基因轉錄本5’ 端未轉譯區形成的偽結結合，使核醣體-mRNA複合體無法進入轉譯前起始階段繼而進行蛋白的合成。目前對於S15蛋白與核醣體-mRNA複合體間的交互作用，以及對其結構的變化會有何影響，使複合體無法進入於轉譯前起始階段的原因則尚未明白。 本篇欲利用單分子螢光共振能量轉移技術去探討rpsO基因轉錄本5’ 端未轉譯區與30S形成轉譯前起始複合物的機制。研究結果發現，此5’ 端未轉譯區確實可形成雙髮夾及偽結結構，與先前研究結果相符。當30S存在時，藉由辨識偽結上裸露的SD (Shine-Dalgarno) 序列並與之結合後就能將RNA下游的二級結構完全解開，同時也發現30S能在雙髮夾結構自發性解開第二個髮夾的瞬間與裸露出的SD序列結合，不過結合後的複合體並不穩定，解開的下游序列傾向摺疊回第二個髮夾結構而迫使30S分離，只有當30S與起始tRNA共同存在才能穩定此核醣體-RNA複合體，並使RNA下游二級解構維持在解開的構型。此外，本篇研究也嘗試探討核醣體S1蛋白在轉譯前起始複合物形成的過程中所扮演的角色。綜合本篇結果可建立rpsO基因轉錄本5’ 端未轉譯區形成轉譯前起始複合物的動態模型，有助於進一步了解轉譯起始階段調控的分子機制。

Escherichia coli ribosomal protein S15 is encoded by the rpsO gene and can regulate its own biosynthesis through the 5’ untranslated region (5’ UTR) of its transcript. The RNA transcript can fold reversibly into a pseudoknot or a double-hairpin conformation. The ribosome only binds to the pseudoknot to initiate the translation reaction. When S15 is in excess in the cell, it will bind to the pseudoknot structure and then block the ribosome from accessing the initeiation site in such a way to stall the translation initiation. How S15 interacts with the mRNA-ribosome complex and influences the conformation of the preintiation complex still remains unclear. In this study, we investigate the dynamic rearrangements of the rpsO 5’ UTR RNA and the formation of preinitiation complexees using the single-molecule fluorescence resonance energy transfer (smFRET) technique. We show that ribosomal subunit 30S can unwind the local downstream structures after binding to the SD sequence of the pseudoknot. The 30S can bind to the double-hairpin while the SD sequence is transiently exposed from the hairpin . In this case, the 30S-mRNA complex is unstable and thus the downstream sequence will refold into the haipin structure again. In the presence of the initiator tRNA, the 30S-mRNA complex will be stabilized and lead to the formation of the preinitiation complex. In addition, we also try to explore the role of ribosomal protein S1 in the process. In summary, we have established a dynamic model of translation initiation based on the rpsO 5’ UTR , which will provide insight into the molecular mechanism of translation initiation.