酵母菌轉譯起始機制的研究

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姓名

唐蕙苓(Huei-Lin Tang) 查詢紙本館藏

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生命科學系

論文名稱

酵母菌轉譯起始機制的研究
(Elucidating a novel translation initiation mechanism for a eukaryotic gene)

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摘要(中)

中文摘要
目前已知在原核及高等真核細胞中，少數的基因可以使用 non-ATG作為轉譯的起始密碼，但在低等真核細胞如酵母菌Saccharomyces cerevisiae卻從未發現過。我們的實驗結果顯示酵母菌只有一個alanyl-tRNA synthetase (AlaRS)的基因 (ALA1)，這個基因可以同時轉譯出細胞質及粒腺體所需的AlaRS。然而在這個基因的5’端卻只有一個ATG起始密碼(即ATG1)，藉由5’ RACE的方法，我們發現ALA1可以轉錄出三條長短不同的訊息RNA，其5’端分別座落在-143，-105和-54核苷酸上。點突變的結果顯示，ALA1以ATG1 為起始密碼轉譯出細胞質的AlaRS，卻以上游的二個non-AUG (即ACG-25和ACG-24)為起始密碼轉譯出較大的粒腺體AlaRS。弁鄐W這兩個ACG都可以用來作為轉譯起始密碼，但是以ACG-25為較主要的起始密碼。用西方轉漬法來測定其蛋白質的表現，也得到了一致性的結果。而這樣的結果也首次證明了在正常生理條件下酵母菌可以使用non-AUG作為轉譯起始密碼，且這項發現不但銜接了由原核生物到高等真核生物在non-AUG使用上的斷層，也讓我們對酵母菌基因表現的調控有了更深一層的認

摘要(英)

Abstract
Initiation of protein translation at non-ATG codons has been shown to occur naturally, although rarely in prokaryotes and high eukaryotes, and never in yeast. In this thesis, we provide strong evidence that a non-ATG codon is used as the alternative translational start site of the yeast gene ALA1, which is the only gene in Saccharomyces cerevisiae coding for alanyl-tRNA synthetase. An in vivo functional assay shows that ALA1 is a bifunctional gene that provides both the cytoplasmic and mitochondrial functions. However, unlike most other bifunnctional genes, which contain two alternative in-frame ATG initiators, there is only one ATG codon, designated ATG1, close to the 5’-end of the ALA1open reading frame. Transcriptional mapping reveals the existence of three overlapping transcripts for ALA1, with 5’ ends at positions –143, -105, -54, respectively, relative to the “A” nucleotide of ATG. Site-specific mutagenesis shows that the cytoplasmic and mitochondrial functions of ALA1 are provided by two distinct protein products: a cytoplasmic form initiated at ATG1 and a longer mitochondrial form initiated at two in-frame non-ATG codons, ACG-25 and ACG-24. These two ACG codons function redundantly in initiation of translation. Either one can be functional in the absence of the other; however, the first ACG codon appears to play a predominant role in protein synthesis. Western blot analysis further confirms the initiator function of these two codons. This appears to be the first example in which a non-ATG codon is used physiologically as a transitional initiator in yeast.

關鍵字(中)

★ non-AUG 轉譯起始密碼

關鍵字(英)

★ alternative transcription and translation
★ leaky scanning
★ non-AUG initiator

論文目次

目錄
目錄……………………………………………………………………………. I
圖、表目錄………………………………………………………………….. III
縮寫檢索表………………………………………………………………….. IV
中文摘要……………………………………………………………………… 1
英文摘要……………………………………………………………………… 2
第一章緒論
I. Aminoacyl-tRNA synthetase (aaRS)的簡介…………………………. 4
1. aaRS的生化function.................................4
2. aaRS的分類……………………………………………………… 5
II. 原核與真核細胞中aminoacyl-tRNA synthetase在轉譯方式
上的差異……………………………………………………………… 5
III. 酵母菌中aaRS轉譯方式的差異…………………………………... 6
IV. 轉譯起始密碼的選擇………………………………………………... 7
1.絕大多數基因使用ATG作為轉譯起始密碼，少數則使用
non-ATG作為轉譯起始密碼……………………………………... 7
2.酵母菌中alanyl-tRNA synthetase的特性……………………….. 8
V. 研究目的………………………………………………………………9
第二章材料與方法
一、使用之菌株、載體及培養基……………………………………… 10
二、製備少量質體DNA……………………………………………….. 11
三、限制酵素切割質體DNA………………………………………….. 12
四、鹼性去磷酸酵素(BAP)處理載體…………………………………. 12
五、接合反應 (ligation)……………………………………………….. 13
六、大腸桿菌之形質轉換(Transformation)………………K……….. 14
七、酵母菌之形質轉換(Transformation)…………………………….. 15
八、5’ RACE (rapid amplification of cDNA ends)…………………... 16
九、Site-Direct Mutagenesis…………………………………………... 18
十、互補試驗(Complementation )……………………………………. 19
十一、蛋白質製備(Protein preparation)……………………………... 20
十二、SDS-PAGE之蛋白質分子量分析……………………………... 21
十三、西方點漬法(Western blotting)…………………………………. 22
十四、蛋白質濃度的定量 (Bradford, 1976)…………………………... 24
第三章結果
I. 一個ALA1基因轉錄多條訊息RNA………………………………. 25
II. 一個ALA1基因轉譯二個蛋白質…………………………………. 26
III. 粒腺體AlaRS使用non-AUG當轉譯起始密碼………………... 27
IV. 用西方吸漬法證明粒腺體AlaRS使用non-AUG當轉譯起
始密碼…………………………………………………………….. 29
第四章討論……………………………………………………………….. 30
第五章參考文獻………………………………………………………….. 32
圖表………………………………………………………………………….. 36
附錄………………………………………………………………………….. 49
圖、表目錄
圖1、Aminoacylation……………………………………………………….. 36
圖2、aaRS在細胞中的function and role…………………….. 36
圖3、one gene two products………………………………………………... 37
圖4、sequence context……………………………………………………… 37
圖5A、ALA1基因的5’序列………………………………………………... 38
圖5B、酵母菌的AlaRS與其它不同物種的細胞質AlaRS N
端序列比對………………………………………………………….. 39
圖6A、以5-FOA培養基測試各種不同的ALA1突變基因
之互補能力………………………………………………………….. 40
圖6B、以YPG培養基測試各種不同的ALA1突變基因之互補能力…… 41
圖7A、以5-FOA培養基測試各種不同的ALA1突變基因之互補能力… 42
圖7B、以YPG培養基測試各種不同的ALA1突變基因之互補能力…… 43
圖8A、ALA1 基因前端序列(-1 ~ -120 bp)作不同的突變之後lexA
基因融合所構築的質體……………………………………………. 44
圖8B、西方點漬法觀察 in vivo中蛋白質的表現……………………….... 45
表1. Class I及Class II 的分類…………………………………………….. 46
表2. Class I及Class II主要特性……………………………………………46
表3、各種不同的ALA1突變基因及其互補能力測試…………………… .47
表4、各種不同的ALA1突變基因及其互補能力測試…………………… .48

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指導教授

王健家(Chien-Chia Wang)

審核日期

2002-6-24

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