啤酒酵母的Spt5 對於第二型RNA聚合酶複合體之結構的影響

Abstract

在真核生物內，轉錄延長因子Spt5對於基因表達扮演重要角色。此外，它也是個多功能因子，它不僅促進了RNA polymerase II（RNAPII or Pol II）從初始到終止階段之間的轉錄作用。也對於人類神經退化性疾病與愛滋病造成影響。於是Spt5-Pol II的研究就非常重要。本實驗室同仁先前對於Spt5-Pol II複合體，使用冷凍電子顯微鏡(cryo-EM)結構，說明Spt5可能會改變Pol II與Spt5-Pol II轉錄複合體的立體構型。因此，我們利用啤酒酵母菌的Spt5與RNA polymerase II，研究Spt5對於Pol II的結構影響。為了克服Spt5產量的要求，我們針對Spt5建立了一個啤酒酵母過量表達系統。首先，我優化了此過量表達系統與開發了一個技術。本技術利用免疫沈澱固定Pol II。另外，我利用size exclusion chromatography做競爭性分析試驗，藉此評估Spt5與Pol II的互動關係。最後，我發現了在缺乏DNA-RNA scaffold結構存在，Spt5會把次複合體Rpb4/7從RNA polymerase II上剔除。最重要的是，此發現對於近一步探討Spt5與RNA polymerase II的互動情形，開啟了新的可能。

In the eukaryotes, transcription elongation factor Spt5 plays important role in the gene expression. Also, Spt5 is a multi-functional factor. It not only facilitates the RNA polymerase II (RNAPII or Pol II) mediated transcription, from initiation to termination, but also has effects on human neurodegenerative disease and AIDS. Thus, the study on Spt5 and Pol II is very important. Our previous cryo-EM structure study of Spt5-Pol II complex suggested that Spt5 could alter the architecture of Pol II transcribing complex. Therefore, we studied the Spt5 effect on the architecture of Pol II using the S. cerevisiae’s Spt5 and Pol II. To overcome the demand on the quantity of Spt5, we construct an over-expressing system of S. cerevisiae to produce Spt5. Here, I optimized the S. cerevisiae over-expressing system for Spt5 and developed an immobilized Pol II assay with immuno-precipitation (IP) and Spt5-Pol II competition assay with size exclusion chromatography allowing for evaluating the interactions between Spt5 and Pol II both from S. cerevisiae. Finally, I discovered that Spt5 may dissociate subcomplex Rpb4/7 from core structure of RNA polymerase II without DNA-RNA scaffold. Last but not least, the findings initiate new possibility