一個雙重功能的酵母菌 tRNA 合成酶之研究

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姓名

桂妤(Yu Kuei) 查詢紙本館藏

畢業系所

生命科學系

論文名稱

一個雙重功能的酵母菌 tRNA 合成酶之研究
(Study of a bifunctional yeast tRNA synthetase of Candida albicans)

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摘要(中)

在 Saccharomyces cerevisiae 中， ALA1 可以同時解碼細胞質與粒線體的 alanyl-tRNA synthetase (AlaRS)。此一基因利用最靠近 mRNA 5’端的 AUG (即 AUG1) 轉譯出細胞質異構型，利用 AUG 1 上游的兩個重複密碼 ACG (-25) 與 ACG (-24) 轉譯出粒線體異構型。本論文中，我們選殖了 Candida albicans 的 ALA1 基因 (CaALA1)，並針對這個基因的蛋白質轉譯及細胞內分布做進一步的研究，我們發現 CaALA1 可以同時取代 S. cerevisiae ALA1 基因的細胞質與粒線體功能，然而其基因表達的方式卻非常不同。首先我們發現 CaALA1 只轉錄一條 mRNA ，其 5’端位於 AUG1 上游的 24 個核苷酸處。再者， CaALA1 是利用其 mRNA 上的 AUG1 轉譯出一個同時具有粒線體與細胞質功能的蛋白質，而利用與 AUG1 相距八個密碼的 AUG9 轉譯出一個只具有細胞質功能的蛋白質異構型。進一步將此細胞的細胞質及粒線體分離，結果發現由 AUG1 做出的蛋白質可同時分布於細胞質及粒線體，相對地，由 AUG9 做出的蛋白質則只分布於細胞質，此結果與功能性分析完全一致。點突變的實驗結果顯示 CaALA1 的轉譯可能是透過 Leaky scanning 的方式進行，因此改變 AUG1 的週邊序列不但會影響 AUG1 的轉譯起始效率，也會影響 AUG9 的效率。據我們所知，這是在酵母菌中發現的第一個例子：一個 tRNA synthetase 可以同時跑到兩個不同的胞器中作用。

摘要(英)

It was previously shown that the cytoplasmic and mitochondrial activities of alanyl-tRNA synthetase of Saccharomyces cerevisiae are provided by two translational products of ALA1, one initiates at the AUG codon closest to the 5’-end of its mRNA transcripts and the other at upstream in-frame redundant non-AUG codons (i. e., ACG(-25) and ACG(-24)). In this thesis, we report the cloning and characterization of a homologous gene from Candida albicans. Functional assays show that this gene can substitute for both the cytoplasmic and mitochondrial functions of ALA1 in S. cerevisiae; however, several points regarding the pattern of gene expression differ from mechanisms used to regulate expression of ScALA1. First, the results of 5’-RACE showed expression of only a single transcript whose 5’-end mapped to nucleotide position -24 relative to ATG1. However, this single transcript codes for two distinct protein isoforms through alternative initiation from two in-frame AUG triplets 8 codons apart. Complementation assays showed that the AUG1-initiated protein can complement for both cytoplasmic and mitochondrial activities of ScALA1, while the AUG9-initiated protein functions only in the cytoplasm. Fractionation assays also showed that the AUG1-initiated protein form can be partitioned in both compartments; however, the AUG9-initiated protein form was exclusively confined to the cytoplasm. Next, we tested whether the mechanism used by CaALA1 to initiate translation was due to “leaky scanning”. Therefore, a series of point mutations were introduced into the sequence context surrounding the first initiator. Results showed that the efficiencies of translation initiation by the two initiators could be influenced by changing the sequence context of AUG1, consistent with the leaky scanning model of translation initiation. To our knowledge, this appears to be the first example in yeast wherein a naturally occurring form of a tRNA synthetase can play roles in both compartments.

關鍵字(中)

★ 雙功能
★ tRNA合成酶
★ 酵母菌

關鍵字(英)

★ Candida albicans
★ bifunctional
★ alanyl-tRNA synthetase

論文目次

中文摘要 I
英文摘要 II
誌謝 III
目錄 IV
圖表目錄 VI
縮寫檢索表 VII
第一章緒論 1
1. Aminoacyl-tRNA synthetase (aaRS) 的簡介 1
2. 原核與真核細胞在轉譯方式上的差異 1
3. 少數的真核細胞 aaRS 只有一個細胞核基因 2
4. 比較 S. cerevisiae 與 Candida albicans 之間 AlaRS 蛋白質表現差異4
5. 真核細胞的轉譯機制4
6. 研究目的5
第二章材料與方法 6
1. 使用之菌株、載體及培養基 6
2. 大腸桿菌勝任細胞的製備與轉型作用 6
3. 酵母菌勝任細胞的製備與轉型作用 8
4. 質體之選殖 9
5. 鑑定 CaALA1 mRNA 的 5’端 10
6. 點突變 (Site-directed Mutagenesis) 13
7. 功能性互補試驗 (Complementation ) —測試細胞質功能 14
8. 功能性互補試驗 (Complementation ) —測試粒線體功能 15
9. 蛋白質製備 (Protein Preparation) 17
10. SDS-PAGE之蛋白質分子量分析 18
11. 西方氏點墨法 (Western blotting) 19
12. 酵母菌粒線體的分離 (Enrichment of mitochondria) 20
第三章結果 22
1. CaALA1 基因序列的分析 22
2. CaALA1 具有細胞質與粒線體雙重功能 23
3. CaALA1 可由不同的轉譯起始點合成兩個蛋白質異構型 24
4. CaAlaRS 在細胞中的分布情形 25
5. 比較 AUG1 與 AUG9 的轉譯起始效率 28
6. 以回報基因分析 CaAlaRS 的合成方式 28
7. 週邊序列影響轉譯起始點的效率 29
第四章討論 32
1. CaALA1 為一個雙重功能基因 32
2. 一個 CaAlaRS 異構型可以同時作用於兩個胞器 33
3. 粒線體基質胜??切割效率的探討 34
4. 酵母菌 Leaky scanning 的探討 35
第五章參考文獻 37
圖表 41
附錄 58

參考文獻

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黃曉芸 (2005) 酵母菌 ALA1 基因轉譯起始機制的研究。中央大學碩士論文

指導教授

王健家(Wang Chien-Chia)

審核日期

2006-7-18

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