出芽酵母Spt5在RNA polymeraseⅡ上的功能與結構的觀察

Abstract

在真核生物中,核糖核酸聚合酶二型是由12個次單元體構築的巨型蛋白質複合體,負責轉錄信使RNA以及小核RNA、小分子RNA,而轉錄因子Spt5對於核糖核酸聚合酶二型轉錄的初始、延長、以及終止階段都扮演了角色。 本篇論文,以出芽酵母過量表達可被半乳糖誘導表現的Spt5,以體外的轉錄實驗驗證了蛋白質的功能性,並利用冷凍電顯技術對於Spt5與核糖核酸聚合酶二型的複合體進行三維結構的組建。在結構影像中我們有意外的發現,本篇首次揭露Spt5與核糖核酸聚合酶二型的結合,會造成其柄狀構型的Rpb4/7自核糖核酸聚合酶二型分離,並且這個現象也在生理狀況被觀測到。 總結來說,本篇成功過量表現有功能性的蛋白質Spt5,其與核糖核酸聚合酶二型的結合造成Rpb4/7的分離,實驗結果可能揭露了一個新的Spt5對於轉錄調控的機制。

In the eukaryotes, RNA polymerase Ⅱ is a large protein complex consist of 12 subunits·It catalyzes the transcription of DNA to pre-mRNA、Small nuclear RNA and microRNA. Transcription Factor Spt5 play roles in RNA polymerase Ⅱ mediated transcription, from initiation to termination. Here, we use Saccharomyces cerevisiae system to overexpress galactose inducible Spt5, and characterized its function and interaction with RNA polymerase Ⅱ in a in vitro transcription system. We use Cryo-EM and 3D-reconstruction to demonstrate structure of Spt5/ RNA polymerase Ⅱ complex. Surprisingly, our structure image rollout Spt5 binding with RNA polymerase Ⅱ will lead stalk-like Rpb4/7 heterodimer dissociate from RNA polymerase Ⅱ, and this event will occur during in vivo transcription. In summary, we successfully overexpress functional protein Spt5.Spt5 bind to RNA polymerase Ⅱ will lead Rpb4/7 dissociate from RNA polymerase Ⅱ, this result may reveal a new mechanism of Spt5 to regulate transcription process 。