酵母菌使用罕見轉譯起始密碼的可能性探討

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姓名

張嘉珮(Chia-pei Chang) 查詢紙本館藏

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生命科學系

論文名稱

酵母菌使用罕見轉譯起始密碼的可能性探討
(Translation initiation from novel initiators in yeast)

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★ 酵母菌valyl-tRNA synthetase附加區段的生物功能之探討	★ 探討酵母菌glycyl-tRNA合成酵素的非傳統生物功能
★ 探討酵母菌Valyl-tRNA synthetase的生化活性	★ 酵母菌轉譯起始機制的研究

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摘要(中)

先前研究已知，在Saccharomyces cerevisiae 中，粒腺體以及細胞質功
能的alanyl-tRNA synthetase (稱為AlaRS)是由ALA1 基因所提供。ALA1 基
因利用最靠近mRNA 5’ 端的ATG (1)轉譯出細胞質所需的AlaRS，再利用
ATG1 上游的二個重複ACG (即ACG (-25)和ACG (-24)) 做出粒腺體異構
型，主要仍以ACG (-25)為主。本篇論文著重於酵母菌中使用non-ATG 作為
轉譯起始密碼相關機制的研究。我們以ALA1 為對象去篩選在自然的情況
下，是否有其它的密碼可以取代原本提供粒腺體功能的轉譯起始密碼
(ACG (-25))，篩選結果發現和高等真核生物一樣，酵母菌可以使用和ATG
只差一個核苷酸的密碼 (ATA, ATU, ATC, GTG, TTG, CTG)作為轉譯起始
點，除此之外，我們也篩選到非常罕見且一般不被認為的轉譯起始點密碼
(AGC, CGC, CAT, CAC)可以取代ACG (-25)。我們利用西方點墨法進一步定
量發現，周圍序列-3 ~ -1 的核苷酸對轉譯效率有非常大的影響；另外我們
發現ACG (-25)下游的一個二級結構對轉譯效率也有非常大的影響，其詳細
機制有待進一步研究。
論文第二部份，著重於酵母菌中valyl-tRNA synthetase (ValRS)附加區段
的研究。我們發現這個附加區段具有非專一性tRNA 鍵結的能力，幫助tRNA
鍵結至valine 的酵素上 (Kd 約為2 μM)，而對此附加區段做部分刪除，例
如Δ32-71，便破壞酵素對tRNA 鍵結的能力及酵素胺醯化的活性。除此之
外，先前研究研究顯示附加區段活化轉錄的活性，而我們發現ValRS 可藉
由附加區段將GFP 送入細胞核中，這些結果顯示ValRS 可能參與細胞核基
因的調控。

摘要(英)

Recent studies in Saccharomyces cerevisiae have shown that ALA1 (coding
for alanyl-tRNA synthetase) initiates the translation of its mitochondrial isoform
from two consecutive ACG triplets, with the first ACG being the more robust. In
the work described here we focuse on the mechanism of non-ATG initiation in
yeast. To explore if any other non-ATG triplets can serve a similar function, the
first and second ACG triplets were replaced by a random triplet and ACC,
respectively, and the resultant constructs were screened for candidates
expressing the mitochondrial activity. We report here that, in addition to ATG,
six common non-ATG initiators (ATA, ATU, ATC, GTG, UTG, and CTG) and
four rare non-ATG initiators (AGC, CGC, CAT, and CAC) can each functionally
substitute for the ACG initiators. Western blot analysis suggests that initiating
activity of the non-ATG initiators is significantly affected by the nucleotides at
their relative positions -3 ~ -1. Furthermore, a putative pseudoknot structure
17-nucleotide downstream of the non-AUG initiation site also appears to be
important for the initiation activity.
A second part of the thesis is focused on the valyl-tRNA synthetase (ValRS)
appended domain (ad) of Saccharomyces cerevisiae. We show here that this
domain is a non-specific tRNA-binding domain (Kd ~ 2 μM) that contributes
significantly to the tRNA-binding activity of the valine enzyme. Thus, even a
small deletion in the appended domain, such as Δ32-71, has a devastating effect
on the enzyme’s tRNA-binding and aminoacylation activities. In addition we
show here that the ad can deliver a green fluorescent protein in to the nucleus,
further reinforcing the notion that ValRS could be involved regulation of nuclear
gene.

關鍵字(中)

★ 酵母菌轉譯機制

關鍵字(英)

★ non-ATG
★ ALA1

論文目次

中文摘要 i
英文摘要 ii
誌謝 iii
目錄 iv
圖目錄 vii
表目錄 viii
第一章緒論 - 1 -
1.1 Aminoacyl-tRNA synthetases (aaRSs)的簡介 - 1 -
1.1.1 aaRS的功能 - 1 -
1.1.2 aaRS 的分類 - 2 -
1.2 原核與真核細胞在轉譯起始上的差異 - 4 -
1.2.1 原核生物的轉譯起始 (translation initiation)作用 -4 -
1.2.2真核生物的轉譯起始作用 - 5 -
1.3Alanyl-tRNA synthetase (AlaRS)的簡介 - 7 -
1.4Valyl-tRNA synthetase (AlaRS)的簡介 - 8 -
1.4.1ValRS的生化特性 - 8 -
1.4.2酵母菌中的ValRS - 9 -
1.5研究目的 - 10 -
第二章材料與方法 - 12 -
2.1菌株、載體及培養基 - 12 -
2.2大腸桿菌勝任細胞的製備與轉型作用 - 13 -
2.2.1大腸桿菌勝任細胞的製備 - 13 -
2.2.2大腸桿菌勝任細胞的轉型作用 (transformation)- 14 -
2.3酵母菌勝任細胞的製備與轉型作用 - 14 -
2.3.1酵母菌勝任細胞的製備 - 14 -
2.3.2酵母菌勝任細胞的轉型作用 - 15 -
2.4質體之選殖 - 15 -
2.5點突變 (Site-directed Mutagenesis) - 16 -
2.6功能性互補試驗 (Complementation)―測試細胞質功能- 17 -
2.7功能性互補試驗 (Complementation)―測試粒腺體功能- 18 -
2.8蛋白質製備 (Protein preparation) - 19 -
2.9SDS-PAGE之蛋白質分子量分析 - 20 -
2.10西方點墨法 (Western Blotting) - 21 -
2.11酵母菌融合蛋白質的表現與純化 - 22 -
2.12胺醯化作用分析 - 24 -
2.13聚丙烯醯胺親和力共電泳 (Polyacrylamide Affinity Coelectrophoresis；PACE) - 26 -
2.14ValRS附加區段的細胞核標的訊號測試 - 27 -
第三章結果 - 29 -
3.1篩選酵母菌中可用的各種轉譯起始點 - 29 -
3.2周圍序列對non-ATG轉譯效率的影響--西方點墨法- 31 -
3.3周圍序列對non-ATG轉譯效率的影響—互補測試 - 32 -
3.4下游二級結構對non-ATG轉譯效率的影響 - 34 -
3.5酵母菌ValRS的附加區段對ValRS胺醯化作用的影響- 35 -
3.6酵母菌ValRS的附加區段會鍵結tRNA - 37 -
3.7ValRS附加區段會將蛋白質送入細胞核 - 38 -
第四章討論 - 39 -
4.1酵母菌中可用的各種轉譯起始點 - 39 -
4.2酵母菌中ValRS附加區段的特性 - 40 -
參考文獻 - 43 -

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謝佳容 ( 2002) 酵母菌valyl-tRNA synthetase附加區段的生物功能之探討。中央大學碩士論文
葉蟬嫻 ( 2003) 探討酵母菌ALA1基因的 non-AUG轉譯機制中央大學碩士論文
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
林明琁 ( 2005) 探討一個真核tRNA合成酶的附加區段之轉錄活化活性。中央大學碩士論文

指導教授

王健家(Chien-chia Wang)

審核日期

2007-7-16

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