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標題: D型反轉錄病毒的CTE RNA和其寄主蛋白間交互作用之分析
作者: 劉豫屏
關鍵字: Constitutive transport elements;Tip-associated protein
出版社: 生物化學研究所
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.
Appears in Collections:生物化學研究所

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