酵母菌HTS1基因轉譯起始機制之研究

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姓名

郭亦亦(Yi-Yi Kuo) 查詢紙本館藏

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生命科學系

論文名稱

酵母菌HTS1基因轉譯起始機制之研究
(Translation initiation of HTS1 in yeast)

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摘要(中)

在酵母菌中有兩套不同的細胞核基因，分別控制細胞質及粒腺體 aminoacyl-tRNA synthetase（aaRS）的合成。這兩套細胞核基因在轉譯完成後，其中一個 aaRS 會留在細胞中進行細胞質蛋白質合成，而另外一個帶有粒腺體標的訊號的 aaRS 則會被送到粒腺體中去作用。然而Saccharomyces cerevisiae HisRS（ScHisRS）只用一個HTS1基因轉譯出兩種異構型酵素，分別在粒腺體與細胞質中進行胺醯化反應。HTS1基因上含有兩個in-frame的ATG，分別為ATG1及ATG21。根據前人實驗的結果發現HTS1基因，會轉錄出兩種mRNA，較長的mRNA包含了AUG1及AUG21，而較短的mRNA只有AUG21。在本實驗中我們選殖了Schizosaccharomyces pombe、Candida albicans和Aspergillus fumigatus的HTS1基因，利用互補性實驗鑑定這些基因的轉譯起始密碼，結果發現它們也有兩個in-frame的AUG，分別做出粒腺體與細胞質酵素。此外當我們比較ScHisRS及大腸桿菌的HisRS序列後，其中ScHisRS較大腸桿菌的N端多了一段34個胺基酸的附加區段，此附加區段並不能活化大腸桿菌GlnRS，因此推測ScHisRS附加區段不能增加E. coli GlnRS對酵母菌tRNAHis結合的親和力。此外，刪除了ScHisRS附加區段對於其細胞質和粒腺體的活性是沒有影響的，所以我們推測在in vivo的情況下HisRS的附加區段對於HisRS的催化活性並非必需的。

摘要(英)

In yeast, there are two sets of aminoacyl-tRNA synthetases, one localized in the cytoplasm and the other in the mitochondria. Most of the mitochondrial tRNA synthetases are encoded by nuclear genes distinct from those encoding their cytoplasmic counterparts. However, some mitochondrial tRNA synthetases are encoded by the same genes that code for their cytoplasmic homologues. For example, the cytoplasmic and mitochondrial histidyl-tRNA synthetases of Saccharomyces cerevisiae (ScHisRS) are encoded by the same nuclear gene, HTS1, through alternative initiation of translation from two in-frame AUG codons. The gene specifies two messages, the longer one with two 5’-end in-frame AUGs and the short one with only the second AUG. In this study, we showed that the HisRS genes of Schizosaccharomyces pombe、Candida albicans and Aspergillus fumigatus have only one gene that also contain two in-frame AUG initiation codons. The mitochodrial and cytosolic forms are translated from the first and second AUG initiation codons, respectively. Besides, we found that ScHisRS had a lysine-rich N-terminal polypeptide extension of 34 residues, which was absent from its E. coli counterpart. Attachment of this appended domain to E. coli glutaminyl-tRNA synthetase did not enhance the enzyme’s tRNA-binding and aminoacylation activity toward yeast tRNAs. Deletion of the appended domain of ScHisRS had little effect on the enzyme’s complementation activity in vivo. These results suggest that the appended domain is not essential for the cytoplasmic and mitochondrial functions of ScHisRS in vivo.

關鍵字(中)

★ 轉譯起始機制
★ 酵母菌

關鍵字(英)

★ translation initiation
★ Histidyl-tRNA synthetase

論文目次

目錄
中文摘要 i
英文摘要 ii
誌謝 iii
目錄 iv
圖目錄 vii
縮寫檢索表 viii
第一章緒論 1
1.1 Aminoacyl-tRNA synthetases（aaRSs）的簡介 1
1.1.1 aaRSs的生化功能 1
1.1.2 aaRSs 的分類 2
1.2 原核與真核細胞在蛋白質合成轉譯方式的差異 3
1.2.1 原核與真核細胞內蛋白質的合成 3
1.2.2少數真核細胞aaRS只有一個細胞核基因 4
1.3 Histidyl-tRNA synthetase（HisRS）的簡介 5
1.3.1 HisRS的生化功能 5
1.3.2 HTS1基因的表現 6
1.4酵母菌aaRS的附加區段 7
1.5研究目的 8
第二章材料與方法 9
2.1使用之菌株、載體及培養基 9
2.2大腸桿菌勝任細胞的製備與轉型作用 10
2.2.1大腸桿菌勝任細胞的製備 10
2.2.2大腸桿菌勝任細胞的轉型作用（Transformation） 11
2.3酵母菌勝任細胞的製備與轉型作用 11
2.3.1酵母菌勝任細胞的製備 11
2.3.2酵母菌勝任細胞的轉型作用（Transformation） 12
2.4質體之選殖 12
2.5點突變（Site-directed Mutagenesis） 13
2.6 hts1剔除株的製備 14
2.7功能性互補試驗（Complementation）：測試細胞質功能 16
2.8功能性互補試驗（Complementation）：測試粒腺體功能 17
2.9蛋白質製備 18
2.10 SDS-PAGE之蛋白質分子量分析 19
2.11西方點墨法（western blotting） 20
2.12酵母菌融合蛋白質的表現與純化 21
第三章結果 24
3.1酵母菌HisRS蛋白質序列的比對 24
3.2不同物種間的互補性實驗 25
3.3 ScHTS1基因的表現 26
3.4利用互補性實驗測試SpHTS1與CaHTS1轉譯起始密碼 27
3.5鑑定AfHTS1轉譯起始密碼 30
3.6 ScHisRS的附加區段是否具鍵結tRNA的能力 32
3.7 ScHisRS附加區段對其細胞質與粒腺體酵素的重要性 33
第四章討論 35
4.1酵母菌HTS1基因的表現 35
4.2 ScHisRS附加區段的特性 37
第五章參考文獻 39
附錄 59
圖目錄
圖一、選擇性轉錄及轉譯機制 43
圖二、酵母菌HisRS蛋白質序列的比對 44
圖三、不同物種間的互補性實驗 45
圖四、利用互補試驗分析野生型及突變種ScHisRS的生長狀態 46
圖五、利用互補實驗以低複製載體測試ScHisRS的生長狀態 47
圖六、SpHTS1的基因序列 48
圖七、利用互補試驗分析野生型及突變種SpHisRS的生長狀態 49
圖八、CaHTS1的基因序列 50
圖九、利用互補試驗分析野生型及突變種CaHisRS的生長狀態 51
圖十、AfHTS1的基因序列 52
圖十一、利用互補性實驗鑑定AfHTS1轉譯起始的密碼 53
圖十二、酵母菌及大腸桿菌 HisRS 的比較 54
圖十三、利用E. coli GlnRS的融合蛋白進行互補性試驗 55
圖十四、利用E. coli GlnRS的融合蛋白進行互補性試驗 56
圖十五、刪除ScHisRS附加區段對細胞質和粒腺體功能的影響 57
圖十六、在高複製載體下利用互補實驗測試刪除ScHisRS附加區段對其功能的影響 58

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指導教授

王健家(Chien-Chia Wang)

審核日期

2008-7-2

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