以單分子螢光共振能量轉移技術觀測S1如何結合mRNA

Abstract

在細菌中，轉譯以30S結合於mRNA 5’ UTR的RBS，並與fMet-tRNAfMet形成複合物，作為起始。 SD sequence的強弱與mRNA 5’ UTR的結構是決定蛋白質合成效率的重要條件，而多數mRNA 5’ UTR會形成各種二級結構，這些結構會造成動力學上的屏障，核醣體就需要先解開結構才能結合在RBS上，進而造成轉譯起始的效率降低。 文獻中指出核醣體蛋白S1能夠作為30S與mRNA接合的平台，其N端與Domain 1 (D 1)和D 2能夠與30S對接，而D 3- D 6則能結合與解開mRNA 5’ UTR結構，使30S能結合在具結構mRNA的RBS上，得以進入轉譯起始。但是S1是如何解開mRNA 5’ UTR結構，且S1能結合RNA的特性與30S能形成轉譯前起始複合物是否有關聯都還不清楚，因此我們藉由將螢光分別標記在S1與RNA上，利用單分子螢光共振能量轉移技術觀測S1解開mRNA的結構。我們發現S1結合RNA時，S1的C端會朝向RNA的上游，並且觀察到有大於一個S1結合在同一RNA的現象。而從實驗室前人的研究中，我們知道S1能夠穩定的結合在髮夾結構的RNA上，與上面的發現相結合，推測當S1結合於RNA時，S1分子之間可能具有類似協同性的特性。另外，在S1的D4和C端上突變後，發現S1結合雙髮夾結構的強度減弱，並且S1間的協同性也較差。從實驗中可以得到結論:S1的C端在結合同一RNA分子的S1間協同性扮演重要的角色，但是若S1的C端突變則對於結合RNA的影響不大。

In Gram-negative bacteria, the ribosomal protein S1 is essential for translation initiation. The multi-domain protein S1 associates with the assembly of small ribosomal subunit (30S) and helps 30S resolve the structure of RNA. However, how S1 unfolds the structure of mRNA in the translation initiation site is still unknown. Here we use single molecule fluorescence resonance energy transfer to study how S1 binds single-stranded mRNA. We labeled a Cy3 dye on the C-terminus of S1 molecule, and observe how S1 binds structured RNA. We show that the C-terminus of S1 is orientated toward the 5’ end of RNA when binding structured RNA, and also observe that there is more than one S1 binding to structured RNA. Previous data showed that S1 binds structured RNA strongly. Thus, we suppose that there is a like cooperation activity between S1 molecules when they bind structured RNA. In addition, we mutate the Domain 4 and the C-terminus of S1 molecule to observe how the S1 binding ability and cooperation change. We find that mutations cause lower binding affinity and lower cooperation between S1 molecules. We conclude that the C-terminus of S1 is crucial for its cooperation and the decreased cooperation between S1 molecules does not significantly disrupt the RNA binding affinity of S1.