章為皓weihau Chung翁依蘋2019-09-04不公開2019-09-042007http://etds.lib.ntnu.edu.tw/cgi-bin/gs32/gsweb.cgi?o=dstdcdr&s=id=%22GN0694420204%22.&%22.id.&http://rportal.lib.ntnu.edu.tw:80/handle/20.500.12235/100562冷凍電子顯微鏡已經成為結構生物學中一個成熟的工具，特別是可以用來解析無法結晶的巨分子蛋白複合物的近原子結構。在真核生物的蛋白複合物結構生物學中，要去定位每一個獨立的蛋白質次單元是一件非常重要的問題。 傳統上，免疫金（約 5~10 nm）的標記技術被利用在標記獨立的次單元，而負染色電顯足以有效造影。然而免疫金技術需針對蛋白質複合體上的每一個次單元體培養單株抗體，已特異標定次單元體的位置，這種作法是相當昂貴且耗費時間的。此外，由於我們需要較高解析度的影像，免疫金的大小過大（約 5~10 nm），不適合較高解析度的次單元體定位，因此我們在此使用較小的奈米金（1.8 nm）進行標定。 人類基本核酸代謝的酵素複合物大部分在酵母菌中可以找到對應之同源蛋白質複合物，我們利用對酵母菌做基因的操控便利，將小段的胜肽插入所要尋找的次單元中，並利用可辨識該胜肽的小蛋白標記奈米金去定位此段胜肽。 在本篇論文中，我們針對RNA聚合酶轉錄因子TFIIF做研究，它是具有三個次單元的轉錄起始因子，我們能夠純化具有這三個次單元的RNA聚合酶（Tfg1、Tfg2、Tfg3），並在每個次單元中插入攜鈣素親和胜肽（Calmodulin binding peptide），再以純化之攜鈣素（Calmodulin）標記奈米金，使標記奈米金的攜鈣素能與攜鈣素親和胜肽結合，達到定位次單元的目的。 一開始，我們成功的達到這樣的結果，並且利用醋酸鈾負染色取得高對比電顯影像，但是後來我們無法從電顯影像分辨真實的奈米金與醋酸鈾奈米顆粒的差別，導致影像處理上變得非常困難。因此，我們必須利用冷凍電子顯微鏡去觀察，才不會有負染色背景的訊號。於是需找到適合的條件，做出乾淨的無序冰，並且取得RNA聚合酶和1.8 nm的奈米金分別的影像。目前我們正利用此項冷凍電子顯微鏡的技術去取得被1.8 nm的奈米金標記RNAP/TFIIF的影像，以達到定位Tfg1、Tfg2、Tfg3的目的。TEM has become a standard tool in structural biology, particularly in elucidating structure of large protein complexes. In the pursuit of structure of eukaryotic protein complexes, it is very challenging to address the location of individual protein subunits before a near atomic structure is obtained that allows tracing of individual amino acids. Traditionally, immuno-gold labeling technique is exploited to label individual subunits and visualized by light stain EM or cryo EM. However, to generate and screen mono-clonal antibody as the carrier of specific labeler is a time consuming and expensive enterprise. Since most fundamental processes carried out by protein complex in eukaryotes can find their counterpart in yeast, we take advantage of the easy of genetic manipulation with yeast to engineer small peptides into the terminus of individual subunit of interests and use label a small protein that can specific recognize the peptide to carry nano-gold cluster. In this study, we choose to focus on the subunit arrangement of TFIIF, a three subunit transcription initiation factor, in the complex formed by RNA polymerase with it. We were able to purify the complexes from strains of which Tfg1, Tfg2, and Tfg3 was fused with a calmodulin binding peptide respectively, clone and express of calmodulin with gold labeling moiety, and form gold labeled RNA polymerase/TFIIF complexes. Initially, we subject such complexes preserved in light stain condition, to EM imaging. However, we found we were not able to separate the true gold signal from the background from metal clusters formed by uranyl salts. Moreover, the reverse contrast make the parent gold signal only visible inside the boundary of protein signal, making image analysis very challenging. Thus, we resort to cryo TEM techniques and at the beginning we were encountering the issue of choice of cryogenic reagents and have finally overcome the problems to find conditions allowing robust imaging of RNA polymerase/TFIIF complex preserved in amorphous ice. We are now applying such cryo TEM technique to image RNA polymerase/TFIF complexes that are specifically labeled with 1.8 nm nano-gold and to pursue 3-D reconstruction of the golded complexes in order to locate Tfg1, Tfg2 and Tfg3.奈米金攜鈣素RNA聚合酶II轉錄因子nano goldcalmodulinRNA polymerase IItranscription factor