臺灣博碩士論文加值系統

根據本實驗室先前之研究發現核醣體上的核醣體蛋白L7，除了參與核醣體的合成外，還主導核醣體與內質網之間的結合作用。本篇論文主要是探討核醣體蛋白L7如何與內質網相互作用及其作用的結合區域。我們運用基因工程建構了一系列核醣體蛋白L7之變異蛋白質，然後利用內質網微粒漂浮實驗（microsome floating assay），証明當L7僅具氨基端前五十個胺基酸的重複序列時，便可與內質網微粒作用結合；反之，缺乏此重複序列之變異蛋白質則喪失了其結合內質網微粒的親和力。因此說明了此重複序列對於結合內質網是有其必要性並且具有專一性。我們也利用生物感測系統（biosensor: IAsys）來偵測其結合動力學上的分解平衡常數（KD），測得重複序列對內質網的結合親和力其數據與核醣體之KD值相類似。並且發現L7氨基端重複序列的個數之多寡對結合內質網微粒的親和力影響不大，但蛋白質至少須含一個或以上的重複序列才具有其結合內質網的能力。此推論也可經由比對多種L7同源蛋白質的初級結構以及此區域二級結構之親水性質的一致性得到佐證。另外，本篇論文也証明了此序列中帶有核集訊號（nuclear localization signal），並不能成功的進入核仁。同時應用生化免疫之技術測得此重複序列雖然屬於核醣體蛋白的一部分，卻不須參與核醣體的合成。此結果間接地說明此重複序列可能是暴露在核醣體的表面而參與核醣體與內質網之間的相互結合。

According to the previous finding of our laboratory, the ribosomal protein L7 is responsible for binding to the ER membrane. In this thesis, I examine the binding region of ribosomal protein L7 and determine the binding affinity to the ER. The results show that the NH2 terminal region of ribosomal protein L7 makes up a tandem repeated sequence and is essential for binding to the ER membrane. Using a biosensor assay, the dissociation equilibrium constant (KD) of the NH2 terminal tandem repeated sequence to bind to the ER is 2.89 x 10-8 M which is similar to the binding affinity of ribosome, suggesting that this sequence involves in the formation of rough ER. In the same assay, no significant difference in binding affinity to the ER is found for modified proteins regardless the number of repeated units, but protein without any repeated unit does not bind, suggesting that the repeated unit alone rather the number of copies is important to bind. This study suggests that the tandem repeated sequence of the ribosomal protein L7 does not itself intrinsically interact with the ribosomal components but externally engages in binding with the ER membrane assisting the ribosome in the formation of rough ER.

英 文 摘 要 …………………………………………………………... 1
中 文 摘 要 …………………………………………………………... 2
緒 論 …………………………………………………………………... 3
材 料 與 方 法 ……………………………………………………... 7
結 果
一、人類核醣體蛋白質L7及其變異蛋白質基因之建構…………… 28
二、人類核醣體蛋白質L7及其變異蛋白質之表現及純化………… 28
三、人類核醣體蛋白質L7及其變異蛋白質之試管內結合內質網
微粒的能力 …………………………………………………… 29
四、人類核醣體蛋白質L7氨基端重複序列與內質網微粒結合之
動力學分析 …………………………………………………... 31
五、核醣體蛋白質L7的氨基端重複序列在真核細胞內與核醣體
的合成之關係 ………………………………………………... 32
六、核醣體蛋白質L7的氨基端重複序列在真核細胞內表現之情
形 ………………………………………………………………… 33
圖 表 ………………………………………………………………… . 36
討 論
一、人類核醣體蛋白質L7基因序列比對之差異 ………………….. 50
二、人類核醣體蛋白質L7在細胞內已知之生化功能 …………….. 50
三、人類核醣體蛋白質L7的氨基端重複序列參與核醣體與內質
網微粒之結合…………………………………………………... 51
四、從同源核醣體蛋白質L7的序列比對證實此氨基端重複序列
對於結合內質網之重要………………………………………... 52
五、人類核醣體蛋白質L7氨基端重複序列之細胞表現 ………….. 53
六、細胞內核醣體與內質網結合之機制…………………………... 54
附 圖 …………………………………………………………………. 56
參 考 文 獻 …………………………………………………………. 59

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