Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/23889
標題: 核醣核酸解旋酵素(RNA Helicase A)與啟動子 的DNA之間交互作用的分析
作者: 平, 趙
Chao, Ping
關鍵字: 核醣核酸解旋酵素
出版社: 生物化學研究所
摘要: RNA helicase A (RHA) 是一個屬於DEAH家族且與Drosophila的MLE蛋白同源的蛋白。它們都是轉錄活化因子,在細胞核中能解開雙股螺旋DNA或RNA並參與轉錄作用及轉譯後後飾的過程。RHA N端的調節區域中有兩個dsRBM (double-stranded RNA binding motif),被認為可以和雙股RNA之間產生交互作用。根據前人的研究指出RHA的N端區域具有與p16INK4a promoter專一性結合的能力,且能調控p16INK4a promoter的轉錄作用。然而已知dsRBM能辨識雙股核醣核酸,主要是與dsRNA的2’氫氧基結合,用此來區辨dsDNA與dsRNA的不同。因此吾人將RHA1-257做一系列的刪除突變株以EMSA的實驗方法了解其不同部位與dsDNA的結合關係,由結果顯示需要全長的RHA1-257才能與p16INK4a promoter產生交互作用;而RHA1-120必須在高濃度下才能與p16INK4a promoter結合。另外本實驗室在探討CTE-54 RNA與RHA之間的結合關係時,意外發現cyclic-AMP responsive element (CRE) DNA也能競爭RNA-protein complex。所以我同樣以EMSA的方法進行分析,結果顯示CRE DNA同樣具有與RHA1-257結合的能力,且最小結合區域位於RHA1-105。而將RHA1-120中dsRBM1上第54胺基酸,一個高度保留的Lysine改為Alanine,使RHA1-120喪失了與CRE DNA結合的能力,顯示dsRBM1的重要性。進一步以競爭實驗發現,這兩個雙股去氧核醣核酸與RHA之間的結合都具有專一性。但RHA如何能專一性與這兩個序列完全不同的dsDNA結合,則待進一步以定點突變或利用Crystallography的方式了解它們之間的結合機制。
RNA helicase A (RHA) is a member of DEAH family and is a homologue of Drosophila MLE protein. Both of them belong to transcriptional activators. RHA is a nuclear enzyme that catalyzes the unwinding of both double-stranded DNA and RNA, and participates in transcription and post-translation processes. There are two dsRBM (double-stranded RNA binding motif) that might provide affinity for dsRNA at the amino terminal regulatory domain of RHA. dsRBM recognizes the ribose 2'-OH group of dsRNA, and is capable of distinguishing dsDNA from dsRNA. Recently RHA was reported to bind to the p16INK4a promoter in a sequence-specific way. Here, we have designed and produced various deletion mutants of the N terminal domain of RHA and used EMSA to analyze their interaction with p16INK4a promoter. The result indicated that regulatory domain of RHA was essential for p16INK4a promoter binding, whereas RHA1-120 binding to the p16INK4a promoter was observed only at high concentration. Unexpected observation of efficient competition of RNA-RHA complex by cAMP responsive element (CRE) DNA suggested direct binding between RHA and CRE DNA. To explore this possibility, I used the same approach to demonstrate that RHA1-257 is capable of CRE DNA binding, and RHA1-105 is indeed the minimal binding domain. The observation that a point mutation (Lys to Ala) introduced into the conserved dsRBM1 of RHA1-120 resulted in the lost of CRE DNA binding ability, suggested that dsRBM1 is also important for interaction with CRE DNA. In the competition assay, both p16INK4a promoter and CRE DNA interact with RHA1-257 with sequence specificity. However, methods of mutagenesis and structure analysis will be needed to uncover the recognition mechanism underlined.
URI: http://hdl.handle.net/11455/23889
Appears in Collections:生物化學研究所

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