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研究生:劉豫屏
論文名稱:D型反轉錄病毒的CTERNA和其寄主蛋白間交互作用之分析
指導教授:張功耀
學位類別:碩士
校院名稱:國立中興大學
系所名稱:生物化學研究所
學門:生命科學學門
學類:生物化學學類
論文種類:學術論文
論文出版年:2001
畢業學年度:89
語文別:中文
中文關鍵詞:Constitutive transport elementsTip-associated protein
外文關鍵詞:Constitutive transport elementsTip-associated protein
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Constitutive transport elements ( CTE ) 是simple retrovirus中的Mason-Pfizer monkey virus ( MPMV )用來將病毒本身的unspliced及partially spliced RNA 運送到細胞核外的一段序列,CTE RNA 本身為具有兩個identical internal loop-- loop A及loop B及一個AAGA bulge的long stem loop 結構。根據之前的研究loop A及loop B可能是病毒RNA在運送到核外時和寄主蛋白發生交互作用的主要區域。到目前為止,已知會和CTE RNA有交互作用,而且可以幫助CTE RNA 運送出細胞核的寄主蛋白有RNA helicase A ( RHA )及Tip-associated protein ( TAP )兩個蛋白。
本研究的主要目的則在於解析CTE RNA與TAP及之間交互作用的關係,以及CTE RNA中的AAGA bulge及internal loop對其本身構形的影響。在EMSA的實驗中發現只有當CTE RNA中的internal loop及AAGA bulge同時存在時,GST-TAP1-372蛋白與CTE RNA才會具有較強的結合力。另外,在Chemical 及Enzymatic structure mapping的實驗中則發現,當CTE RNA缺乏AAGA bulge時會使其構形產生改變。CTE RNA與GST-TAP1-372蛋白作用後,利用lead acetate作foot-printing時,則更進一步發現在CTE RNA中鄰近AAGA bulge的區域會被GST-TAP1-372蛋白保護住,因此CTE RNA中與TAP蛋白產生交互作用的區域可能包含有AAGA bulge。

The constitutive transport elements ( CTE ) of Mason-Pfizer monkey virus ( MPMV ) are cis-acting element that promote nuclear export of unspliced and partially spliced pre-mRNAs. The CTE forms a long stem loop structure that contains two identical internal loop — loop A and loop B, and a AAGA bulge. The primary sequences in the loop regions of the CTE are important for its function, suggesting that they may contain binding sites for cellular proteins involved in RNA export. Two cellular proteins, RNA helicase A ( RHA ) and Tip-associated protein ( TAP ) were identified as cellular CTE binding factors.
This study is aimed to explore the interaction between CTE RNA and TAP protein, as well as the influence of conformation of CTE on TAP binding specificity. We found both internal loop and AAGA bulge of CTE RNA were required for its high affinity. We also found the conformation of internal loop of CTE RNA will change when lose the AAGA bulge was deleted using the chemical and enzymatic structure mapping. Finally, the foot-printing data reveals part of AAGA bulge of CTE RNA is protected by the TAP . Therefore both internal loop and AAGA bulge of CTE RNA are required to maintain a specific conformation of CTE RNA for it recognition by TAP.

圖次 3
中文摘要 4
英文摘要 5
前言 6
材料與方法 12
一、實驗材料 12
二、實驗方法
1. 將human TAP 1-372構築於pGEX4T-1表現載體 12
2. Human TAP1-372 protein之表現與純化 13
3. 以試管內轉錄作用製備不同形式之CTE RNA 13
4. RNA之5’端γ-32P標定 14
5. RNA之核酸酵素結構分析法( Enzymatic RNA structure
mapping ) 15
6. RNA之化學藥劑結構分析法( Chemical RNA structure
mapping ) 16
7. TAP 1-372與不同形式之CTE RNA結合能力之分析
( EMSA ) 18
8. TAP 1-372與CTE RNA之足跡圖譜分析法
( Foot-printing assay ) 18
結果 21
一、GST-TAP1-372蛋白的表現與純化 21
二、CTE RNA中的bulge及internal loop對與TAP
蛋白結合專一性之影響 24
三、CTE RNA中的bulge及internal loop對其本身
構形穩定度之影響 27
四、CTE 44 RNA中與GST-TAP1-372產生交互作
用的區域 33
討論 36
附錄一 39
附錄二 40
參考文獻 41

參考文獻
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