博碩士論文 93224002 詳細資訊




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姓名 黃曉芸(Hsiao-Yun Huang)  查詢紙本館藏   畢業系所 生命科學系
論文名稱 酵母菌ALA1 基因轉譯起始機制的研究
(Translation initiation of ALA1 in yeast)
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摘要(中) 中文摘要
在Saccharomyces cerevisiae 中,細胞質及粒線體功能的alanyl-tRNA
synthetase (簡寫為AlaRS) 是由ALA1 基因所提供。ALA1 基因利用最靠近
mRNA 5’ 端的AUG1 轉譯出細胞質的異構型,利用AUG1 上游的二個重複
ACG (即ACG-25 和ACG-24) 作出粒線體異構型。在本實驗中我們選殖了
Candida albicans 唯一負責alanyl-tRNA synthetase 的基因 (即CaALA1) ,經
由物種間的功能性基因互補測試,我們發現CaALA1 可以取代S. cerevisiae
ALA1 的細胞質及粒線體功能。然而,不同於S. cerevisiae 的轉譯起始機制,
CaALA1 是利用第一個AUG (即AUG1) 為起始密碼,作出一個同時具有粒線
體及細胞質功能的蛋白質,當AUG1 點突變後,由於失去蛋白質的起始作用,
因此失去粒線體蛋白質的功能,但是CaALA1 可以利用第二個AUG (即AUG9)
為起始密碼,作出細胞質的蛋白質。接著我們利用細胞質功能的valyl-tRNA
synthetase 為回報基因進一步找出其粒線體標的訊號的範圍為包含N端第1 ~
42 個胺基酸,此外AUG1 所做出的前導序列 (胺基酸1 ~ 42) 可以有效地將
回報基因帶到粒線體中。
在本論文的第二部分研究則是著重在進一步探討粒線體ScAlaRS 的細
胞內傳輸機制及功能。藉由功能性互補試驗、點突變、及西方氏點墨法的測
試,我們發現AlaRS 的標的訊號不只包括Met1 上游的前導序列 (胺基酸-25
~ -1),尚涵括了Met1 下游的18 個胺基酸。只有前導序列或胺基端18 個胺
基酸只能將小部份ScAlaRS 運送到粒線體內作用。從ACG-25 起始的蛋白質
會全部運送到粒線體中作用;反之從AUG1 起始的蛋白質幾乎全部都存在於
細胞質中。值得注意的是,AlaRS 的功能除了取決於其標的訊號的長度,也
受到其蛋白質表現量多寡的影響。
摘要(英) It was previously shown that the cytoplasmic and mitochondrial activities of
alanyl-tRNA synthetase of Saccharomyces cerevisiae are provided by two distinct translational
products of ALA1, one initiated 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). We report here the cloning and characterization of the only alanyl-tRNA synthetase
gene of Candida albicans (designated as CaALA1). Cross-species complementation assays
suggest that CaALA1 can substitute for both the cytoplasmic and mitochondrial functions of
ALA1 in S. cerevisiae. However, unlike the case in S. cerevisiae, both of these two activities are
provided a single primary translational product of CaALA1 that is initiated from the first AUG
codon (designated as AUG1) on its coding sequence. When the first AUG codon is inactivated
by point mutations, the mitochondrial function of this gene is impaired, presumably due to
inability to initiate protein synthesis, while its cytoplasmic function can be rescued by
alternative initiation at a downstream AUG codon, AUG9. Functional mapping using the
cytoplasmic form of valyl-tRNA synthetase as the passenger protein identifies the first 42
amino-terminal residues of the Candida protein as the mitochondrial targeting signal. In
addition, evidence shows that an exclusive mitochondrial targeting signal is translationally
initiated from the first AUG initiator.
In the second part of this thesis, we focus on the transport mechanism and physiological
function of mitochondrial ScAlaRS. The results of functional tests, site-directed mutagenesis,
and western blotting all suggest that the mitochondrial targeting signal of ScAlaRS includes
not only the leader peptide but also the first eighteen amino acids downstream of Met1. Neither
the leader peptide nor the eighteen-residue peptide can function as a transit signal per se. By
western blotting assays, our data shows that the ACG-25-initiated proteins are fully translocated
into the mitochondria, and the AUG1-initiated proteins are almost completely retained in the
cytosol. These results confirm our previous observations that cytoplasmic and mitochondrial
activities of ScALA1 are provided by two distinct translational products. In addition, partition
of ScAlaRS is determined by both its leader peptide and expression levels.
關鍵字(中) ★ 雙功能基因 關鍵字(英) ★ bifunctional gene
論文目次 目 錄
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ALA1基因表現差異 3
5. 粒線體標的訊號的特性 4
6. ScAlaRS的粒線體標的訊號 4
7. 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 7
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. 點突變 (Site-directed Mutagenesis) 10
6. 功能性互補試驗 (Complementation ) ―測試細胞質功能 11
7. 功能性互補試驗 (Complementation ) ―測試粒線體功能 12
8. 蛋白質製備 (Protein Preparation) 13
9. SDS-PAGE 之蛋白質分子量分析 14
10. 西方氏點墨法 (Western blotting) 15
11. 酵母菌粒線體的分離 (Enrichment of mitochondria) 16
第三章 結果………………………………………………………………... 18
1. CaALA1 與ScALA1 基因序列比對相似度高 18
2. CaALA1 同時具有細胞質及粒線體功能 19
3. 鑑定CaALA1 的轉譯起始密碼 19
4. AUG1 密碼所起始的蛋白質同時具有細胞質及粒線體活性 20
5. 測定CaAlaRS 在胞器間的分佈 21
6. 利用西方氏點墨法分析CaAlaRS 蛋白質的表現量及大小 22
7. 解析CaAlaRS 的粒線體基質胜肽酶切割位 23
8. 利用回報基因的方式找出CaAlaRS 的粒線體標的訊號 23
9. 利用回報基因的方式找尋決定CaAlaRS 細胞內分佈的區段 24
10. 測定ScAlaRS 粒線體標的訊號的活性 25
11. ScAlaRS 的功能取決於標的訊號的長度及蛋白質表現量 25
12. 探討ScALA1 的轉譯起始機制 26
13. 利用西方氏點墨法證實ScAlaRS 的異構型及胞內分佈 27
14. 利用ScAlaRS-LexA 融合蛋白測定ScALA1 的轉譯機制 28
第四章 討論…………………………………………………………………. 30
1. CaALA1 為一個雙功能基因 30
2. 同一個CaAlaRS 異構型可同時作用於兩個胞器 31
3. CaAlaRS 粒線體標的訊號的特性 32
4. ScAlaRS 粒線體標的訊號的特性 33
5. 酵母菌leaky scanning 機制的探討 33
第五章 參考文獻……………………………………………………………. 35
圖表……………………………………………………………………………. 39
附錄……………………………………………………………………………. 60
圖表目錄
表一、 aaRS催化的胺醯化作用由兩個連續的步驟所組成 39
表二、選擇性轉錄及轉譯機制 40
表三、三種低等真核酵母菌的親緣關係 41
表四、first AUG rule與leaky scanning 42
圖一、Candida albicans ALA1的基因序列 43
圖二、PCR放大及選殖C. albicans的ALA1基因 44
圖三、CaALA1同時具有細胞質及粒線體功能 45
圖四、改變AUG1密碼破壞粒線體CaAlaRS的活性 46
圖五、AUG1作出的CaAlaRS同時有粒線體及細胞質活性 47
圖六、利用ADH 啟動子表現CaALA1 以觀察蛋白質表現情形 48
圖七、利用西方氏點墨法分析CaAlaRS-LexA融合蛋白的表現情形 49
圖八、CaAlaRS的蛋白質N端序列 50
圖九、利用回報基因找出CaAlaRS的粒線體標的訊號 51
圖十、CaAlaRS的粒線體標的訊號是完整且有效率的 52
圖十一、ScValRS的粒線體標的訊號可將CaAlaRS帶入粒線體中作用 53
圖十二、S. cerevisiae ALA1的基因序列 54
圖十三、利用ADH啟動子大量表現ScAlaRS的部分粒線體標的訊號 55
圖十四、利用ADH啟動子大量表現不同長度的ScAlaRS粒線體標的訊號 56
圖十五、利用西方氏點墨法分析ScAlaRS的細胞內分佈 57
圖十六、利用西方氏點墨法分析ScAlaRS-LexA融合蛋白的可能轉譯起始點58
圖十七、lexA mRNA 5’ 端的可能二級結構 59
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碩士論文
指導教授 王健家(Chien-Chia Wang) 審核日期 2005-7-13
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