研究生: |
陳謙 Chen, Chien |
---|---|
論文名稱: |
利用單分子螢光共振能量轉移技術探討RRM1具序列特異性的解旋能力 Sequence-specific unwinding activities of RRM1 revealed by single-molecule FRET spectroscopy |
指導教授: |
李以仁
Lee, I-Ren |
學位類別: |
碩士 Master |
系所名稱: |
化學系 Department of Chemistry |
論文出版年: | 2019 |
畢業學年度: | 107 |
語文別: | 中文 |
論文頁數: | 74 |
中文關鍵詞: | 單分子螢光共振能量轉移 、TDP-43 、DNA 結合力 、解旋力 、dTG 重複序列 |
英文關鍵詞: | single-molecule fluorescence resonance energy transfer(smFRET), TDP-43, DNA binding, double-strand unwinding, dTG repeat sequence |
DOI URL: | http://doi.org/10.6345/NTNU201900237 |
論文種類: | 學術論文 |
相關次數: | 點閱:160 下載:0 |
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TAR 結合蛋白43 (TDP-43) 在真核生物細胞中的轉錄和轉譯過程中起著遺傳調控中有著重要的作用。非正常功能的 TDP-43 在人體內會形成類沉澱蛋白,此有毒物質生成漸凍人症 (amyotrophic lateral sclerosis, ALS) 以及腦前側顳額硬化症 (Frontotemporal lobar degeneration, FTLD)。過去研究發現,TDP-43 可作為一種 RNA/DNA 的結合蛋白 (binding protein),其特定去結合 UG 的重複序列,尤其是位在靠近 3’ 端的囊性纖維化跨膜傳導調節因子 (Cystic fibrosis transmembrane conductance regulator, CFTR) 基因的第九段外顯子 (exon 9) 交接處;同時,TDP-43 具有 RNA 護伴蛋白 (chaperone) 的特性,能夠將雙股螺旋的 RNA 進行解旋 (unwinding)。TDP-43 的結構主要由 RRM1 (RNA recognition motifs)、RRM2、N-末端 (N-terminal domain, NTD) 以及富含甘胺酸 (glycine) 的 C-末端。其中,多項研究指出 RRM1 對於特定 DNA/RNA 序列有較高的結合能力。在此,我們利用單分子螢光共振能量轉移 (single-molecule fluorescence resonance energy transfer, smFRET) 技術來探討 RRM1 解旋 (unwinding) 雙股螺旋 DNA 的能力。實驗結果顯示 RRM1 將雙股螺旋的 TG:CA 重複序列解旋後,會持續停留在原 DNA 試樣上,藉由動力學分析得知解旋的速率決定步驟對 RRM1 的濃度為二級反應,顯示解旋需要兩個 RRM1 同時作用。當我們將 TG 重複序列增長時,RRM1 對 TG 雙股螺旋解旋的出現兩種相互競爭的反應路徑:分別為逐步 (stepwise) 以及直接 (direct) 路徑,且受 RRM1 濃度所調控。而當我們將序列接上非 TG 重複序列時,則沒有觀察到明顯的解旋現象,因此可推斷 RRM1 對序列具有選擇性。
TAR binding protein 43 (TDP-43) plays an important role in genetic regulation during transcription and translation in eukaryotes cells. Malfunction of TDP-43 causes amyotrophic lateral sclerosis (ALS) and Frontotemporal lobar degeneration (FTLD). TDP-43 is primarily an RNA/DNA binding protein with preferential binding to tandem UG sequences located specifically on the 3’- CFTR exon 9. Meanwhile, TDP-43 also possesses RNA-chaperone activity and the ability to unwind double-strand RNA. TDP-43 consists of RRM1, RRM2, and N-/C-terminal domains. Among them, RRM1 shows significantly higher affinity to TG/UG repeats. Here, we utilized single-molecule fluorescence resonance energy transfer (smFRET) spectroscopy to investigate the unwinding activities of RRM1. We observed that RRM1 unwinds double-stranded TG:CA hexa-repeat and stalls on the assay. The observed pseudo-second-order unwinding kinetics suggest a dimeric rate-determining step during the RRM1 unwinding. When a longer TG repeat sequence was used, two competing pathways, stepwise and direct unwinding, were observed with RRM1 concentration dependence. With the repeat sequence connected to non-TG repeat sequences, no unwinding activity was observed, suggesting a strong sequence-specific unwinding ability of RRM1.
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