以單分子螢光共振能量轉移技術探討螢光標定核醣體蛋白bS1如何結合至mRNA 5’端未轉譯區域

Abstract

原核生物的轉譯作用於核醣體進行，然而多數mRNA 的5’端未轉譯區域(5’UTR)容易形成二級結構，可能使核醣體次單元30S無法順利辨認mRNA的轉譯起始序列並正確的結合至mRNA，因而造成轉譯效率下降。核醣體蛋白bS1做為連接30S與mRNA的橋樑，其N端能與30S接合，C端則結合至mRNA，在結構上具有高度靈活性。另外在先前研究中發現bS1具有解旋酶活性，其能夠協助30S解開mRNA的二級結構，促進轉譯起始進行。然而目前對於bS1結合至mRNA的過程及bS1自身構形變化尚未有深入探討。在本研究中，我們製備螢光標定的bS1 (Cy3-S1)與已知5’UTR會形成二級結構的mRNA，以單分子螢光共振能量轉移技術觀測bS1結合至mRNA時彼此間的交互作用。 在研究結果中，我們發現單一個bS1結合到mRNA的方向性為N端朝向mRNA下游，且也有觀察到兩個bS1結合至mRNA的情形，其中bS1結合至mRNA的順序可能為兩個bS1同時結合亦或是一前一後，在此情況下，第二個bS1的結合至mRNA速率會提高許多，可能是因為bS1彼此之間具有協同性。另外，在只有單一個bS1存在的情況下，bS1仍可以穩定結合至mRNA，並解開mRNA的二級結構。綜合以上實驗結果，我們對於bS1與mRNA之間交互作用的分子機制有了更進一步的了解，有助於進一步探究bS1在轉譯起始的生物功能。

The process of translation in prokaryotes is catalyzed by the ribosome. However, most mRNA 5’ untranslated regions (5’UTRs) tend to form secondary structures, which prevent the 30S ribosomal subunit from properly recognizing the translation initiation site and binding to the mRNA, leading to decreased translation efficiency. The ribosomal protein bS1 acts as a bridge between the 30S subunit and mRNA, with its N-terminus binding to the 30S subunit and its C-terminus binding to mRNA, and it has a high degree of structural flexibility. In previous studies, bS1 has been found to possess helicase activity, which can help the 30S subunit unwind secondary structures of mRNA and facilitate the initiation of translation. However, the process of bS1 binding to mRNA and the corresponding conformational changes of bS1 itself has not been thoroughly investigated. In this study, we prepared fluorescence-labeled bS1 (Cy3-S1) and mRNA from known 5’UTR secondary structures and used single-molecule fluorescence resonance energy transfer (smFRET) technology to observe the interactions between bS1 and mRNA. We found that the orientation of bS1 binding to mRNA is with the N-terminus facing the downstream of mRNA. In addition, two bS1 molecules are also observed to bind to one mRNA, in a simultaneous or sequential manner. In the sequential case, the binding rate of the second bS1 is significantly increased, possibly due to the binding cooperativity between bS1 molecules. Additionally, even with only a single bS1, it can still stably bind to mRNA and unwind the secondary structures. Based on the above experimental results, we have gained a deeper understanding of the interaction mechanism between bS1 and mRNA 5’UTR, which will shed light on the biological function of bS1 in translational initiation.