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標題: | Rta參與EB病毒BRLF1-BZLF1雙基因mRNA轉譯起始之機制 Involvement of Rta in the Translational Initiation of BRLF1-BZLF1 Bicistronic mRNA of Epstein-Barr Virus |
作者: | Chun Yin 殷群 |
指導教授: | 張麗冠 |
關鍵字: | EB病毒,Rta蛋白質,轉譯起始,核醣體,18S rRNA, Epstein-Barr virus,Rta,translational initiation,ribosome,18S rRNA, |
出版年 : | 2016 |
學位: | 碩士 |
摘要: | Epstein-Barr virus (EBV)會表現兩個極早期蛋白質Rta和Zta,這兩個蛋白質能幫助病毒進入溶裂期以及早、晚期基因的表現。其中,Zta除了可以由BZLF1單基因mRNA轉譯而來,亦可以由BRLF1-BZLF1雙基因mRNA (RZ-mRNA)的下游開放閱讀框轉譯出來。過去的研究發現Rta可能會幫助40S核醣體的轉移並結合於RZ-mRNA中間序列(IR)上特定的序列-region I,使得40S核醣體能繼續掃描RZ-mRNA,直到辨識到BZLF1的轉譯起始碼並進行轉譯。此外,由核苷酸序列的比對發現region I與18S rRNA的helix 36其中一股的序列互補,而過去研究發現此種序列互補關係對BZLF1基因的轉譯極為重要,因此推測Rta可能與18S rRNA、region I結合,並幫助40S核醣體與IR序列的結合以促進轉譯的起始。本篇研究以in vitro RNA-protein pull-down的方式發現Rta能與18S rRNA的helix 36以及region I的RNA序列結合,而蔗糖密度梯度分層的結果顯示Rta與各個核醣體次單元有共同的分布;此外,本研究也進一步探討Rta是否會影響轉譯起始因子或40S核醣體與region I的結合關係,結果發現雖然RPS6、eIF4A以及eIF4G可以與region I序列結合,但Rta並不會顯著地增加這些蛋白質與region I序列的結合量,說明Rta與18S rRNA或region I序列的結合可能有其他目的。由目前的結果推測region I可能是Rta、RPS6、eIF4A和eIF4G與IR序列結合的位置,而Rta跟核醣體與region I序列皆可能有結合關係。 Epstein-Barr virus (EBV) encodes two immediate-early proteins, Rta and Zta, which promote the lytic activation, and regulate the expression of early and late genes of EBV. The Zta protein can be translated from the BZLF1 monocistronic mRNA or from the downstream open reading frame (ORF) of BRLF1-BZLF1 bicistronic mRNA (RZ-mRNA). Our previous studies demonstrated that the Rta protein could promote the binding of the 40S ribosome to a specific region, called region I, in the RZ-mRNA intercistronic region (IR). Furthermore, the result of sequence alignment revealed that region I was complementary to the helix 36 (n.t. 1484 to 1528) in the 18S rRNA. The sequence complementarity between the IR and 18S rRNA has been found to be critical for the translation of the downstream BZLF1 ORF, suggesting that Rta can bind to 18S rRNA and region I, and facilitate the translational initiation. This study found that Rta was able to bind to not only 18S rRNA but also region I by in vitro RNA-protein pull-down assay. In addition, the Rta proteins were in the same fractions with the ribosomal subunits using sucrose gradient separation. The interactions of region I with RPS6, eIF4A, and eIF4G were also observed in the presence or absence of Rta. Overall, our data indicate that Rta might bind to the RZ-mRNA IR via region I with ribosome and the translation initiation factors. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/78203 |
DOI: | 10.6342/NTU201601106 |
全文授權: | 有償授權 |
顯示於系所單位: | 生化科技學系 |
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