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姓名 唐蕙苓(Huei-Lin Tang)  查詢紙本館藏   畢業系所 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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