利用螢光共振能量轉移技術定位真核轉錄延長複合體

Abstract

RNA聚合酶II具有合成mRNA的功能，在真核細胞中扮演重要的角色。RNA聚合酶II之轉錄延長複合體的X-ray晶體繞射結構已達3.3Å解析度，但由於mRNA具有彈性易擺動，因此轉錄出的mRNA離開RNA聚合酶II之轉錄延長複合體的正確出口途徑，至今仍不是很清楚。 在本文的研究中，利用一種非傳統螢光標定方法來標記RNA聚合酶II次單元，並設計兩種不同長度的RNA，在RNA 5’標記上配對螢光染劑，如此便可測量RNA與RNA聚合酶II次單元之間的距離，推導出轉錄後mRNA的出口路徑。從膠內螢光共振能量轉移技術的結果確定RNA聚合酶II轉錄延長複合體的存在，並成為支持單分子螢光技術實驗結果的另一項證據。 尤有進者，我們成功地建立本實驗室之單分子螢光技術，並利用其可以測量到10-10m解析度之特質，發現Rpb3與不同長度RNA測量螢光轉移共振能量，符合膠內螢光共振能量轉移的結果。此實驗結果在研究RNA的出口途徑之問題上，有十分顯著之意義。

Together with six general transcription factors and promoter DNA, yeast RNA polymerase II (RNA pol II) forms the core enzyme responsible for the synthesis of messenger RNA from genome DNA, a crucial role in eukaryote. The atomic structures of RNA polymerase II elongation complex have been resolved by using X-ray crystallography technology, but the nascent RNA exit route on the complex remains unknown. Fluorescence resonance energy transfer (FRET) is a physical tool that can measure intramolecular or intermolecular distance to nanometer precision. In this work, we use a novel strategy that enables us to measure the distance between 5’ end of designed RNA primers and any subunits in the RNA pol II elongation complex. We have successfully determined the position of 5’ end of the synthesized 10mer and 17mer RNA primers and thus mapped the exiting route of RNA from RNA pol II. Furthermore, we successfully used the in gel fluorescence resonance energy transfer (gelFRET) assay to demonstrate the formation of RNA pol II elongation complex. We further use smFRET to measure the distance between 5’ RNA and c-terminal of Rpb3. Our finding that 10mer RNA assumes higher FRET with the c-terminal of Rpb3 than 17mer RNA suggests the hypothesis RNA exits through a channel toward Rpb7 instead of towards Rpb8.