酵母菌GRS1基因的轉譯起始機制之研究

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姓名

張光容(Kuang-Jung Chang) 查詢紙本館藏

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論文名稱

酵母菌GRS1基因的轉譯起始機制之研究
(Mechanism of translation initiator in yeast GRS1)

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摘要(中)

中文摘要
GRS1是目前酵母菌（Saccharomyces cerevisiae）中唯一已知具功能的Glycyl-tRNA synthetase（GlyRS）基因，雖然在此基因的5`端只有一個ATG轉譯起始點，它卻能同時提供細胞質及粒腺體所需的GlyRS活性。我們的實驗結果顯示，GRS1基因可以同時轉譯出兩個異構型蛋白質：一個轉譯自ATG1，另一個則轉譯自上游相同讀碼框的non-ATG密碼（即TTG -23）；前者作用於細胞質內，後者則作用於粒腺體中。利用5’RACE（rapid amplification of 5’ cDNA ends）與西方點漬法的分析，我們發現這兩個異構型蛋白質是利用leaky scanning的方式，由同一條mRNA上的兩個不同轉譯起始點轉譯而來，這條mRNA的5`端位在相對於ATG1的上游核苷酸-88的位置。雖然這兩種異構型蛋白質有幾乎相同的序列，但因為存在的胞器不同，故在功能上不能互相取代。此外，在融合蛋白的實驗當中，我們發現GRS1的前序列可以解碼出一段胜肽鏈，進而將一個原本屬於細胞質的蛋白質送入粒腺體中，同樣的，GRS1的細胞質異構型蛋白質也可在接上一個外來的粒腺體標的訊號後被送入粒腺體中，這些結果表示GRS1的前序列本身即可解碼出一個完整的粒腺體標的訊號。
在本論文的第二部分，我們著重於酵母菌中使用non-ATG作轉譯起始點的相關機制。利用回報基因分析（reporter gene assay），我們發現兩個並列的non-ATG（ACG-25ACG-24）比一個non-ATG的轉譯效率高，並可取代回報基因的ATG起始點，這是一個從來沒有被發現過的全新機制，表示酵母菌甚或真核細胞可以利用兩個並列的non-ATG來增強原本較差的轉譯起始效率。此外，我們發現在GRS1 TTG-23的下游伴隨一個穩定的RNA二級結構，當我們在此轉譯起始點與二級結構之間加入一段序列時，TTG-23便無法被有效辨識，表示non-ATG存在的位置是被辨識的關鍵因素。另一方面，我們利用隨機突變的方式來篩選中任何可以充當轉譯起始點的密碼，結果發現酵母菌其實跟高等生物一樣可以使用多種跟ATG只差一個核苷酸的non-ATG密碼作為轉譯起始點，但讓人驚訝的是，我們的突變篩選中發現”AGC”竟然也可以當作一個轉譯起始點，據我們所知，這將是第一個在生物中發現的例子：一個跟ATG相差兩個核苷酸的密碼竟可以當作一個轉譯起始點。

摘要(英)

ABSTRACT
GRS1 was previously identified as the only gene coding for glycyl-tRNA synthetase activity in the yeast Saccharomyces cerevisiae. Evidence presented here shows that two distinct protein isoforms are generated from this gene: a short cytoplasmic form, which is translationally initiated at the putative ATG initiator (i.e., ATG1), and a longer mitochondrial form, which is initiated at an upstream in-frame non-ATG codon (i.e., TTG-23). A reverse transcription approach in conjunction with Western blot analysis suggests that the isoforms are translated via leaky scanning from a single transcript of this gene, which has its 5’-end located at position –88 relative to ATG1. Although the isoforms have essentially the same polypeptide sequence, they cannot substitute for each other because of different localization. A domain fusion study suggests that the leader peptide of the mitochondrial isoform can direct a cytoplasmic passenger into mitochondria, while the cytoplasmic form of glycyl-tRNA synthetase can be converted into a mitochondrial protein by fusion of a heterologous signal peptide, suggesting that it is the nature of the leader peptide that is responsible for the subcellular localization of the isoforms.
A second part of the thesis is focused on the mechanism of non-ATG initiation in yeast. Using a reporter gene assay, we show that redundant non-ATG codons have a higher efficiency of translation initiation than a single non-ATG codon at both qualitative and quantitative levels. Even more remarkably, redundant non-ATG codons can substitute for the initiating activity of the ATG initiator of the reporter gene. Thus, the results present a novel mechanism by which the initiating activity of a weak start site can be significantly improved. As in many cases of non-ATG initiation in higher eukaryotes, a stable RNA secondary structure that is predicted to enhance the initiating activity of the non-ATG initiator is found downstream of the GRS1 TTG-23. Introduction of a short sequence between TTG-23 and the secondary structure impairs its initiating activity, suggesting that the distance between the non-ATG initiator and secondary structure is critical for recognition of the weak start site. Our mutagenesis study further shows that, as in higher eukaryotes, many non-ATG codons that differ from ATG by just one nucleotide can be used as translation initiators in yeast. Most surprisingly, an AGC codon, which differs from ATG by two nucleotides, is also functional as an initiator under the conditions used. To our knowledge, this appears to be the first example where a non-ATG triplet that differs from ATG by two nucleotides can still serve as a translation start site.

關鍵字(中)

★ 酵母菌轉譯機制

關鍵字(英)

★ non-ATG
★ GRS1

論文目次

目錄 I
圖、表目錄 III
縮寫檢索表 VI
英文摘要 1
中文摘要 3
第一章緒論
I. Aminoacyl-tRNA synthetase (aaRS)的簡介 5
1. aaRS的生化功能 5
2. aaRS的分類 6
II. 真核細胞內的aminoacyl-tRNA synthetase（aaRS） 7
1. 真核細胞內的aaRS 7
2. 少數的真核細胞aaRS只有一個細胞核基因 7
III. 轉譯起始密碼的選擇 8
1. 少數的高等真核細胞基因可以使用non-ATG作為
轉譯起始密碼 8
2. 酵母菌中GRS1基因的轉譯機制 9
IV. 研究目的 10
第二章材料與方法
一、使用之菌株、載體及培養基 12
二、大腸桿菌之形質轉換(Transformation) 13
三、酵母菌之形質轉換(Transformation) 14
四、質體之建構 16
五、找出GRS1 mRNA的5`端 18
六、Site-Direct Mutagenesis 20
七、互補試驗(Complementation ) 20
八、蛋白質製備(Protein preparation) 22
九、SDS-PAGE之蛋白質分子量分析 23
十、西方點漬法(Western blotting) 25
第三章結果
I. 一個GRS1基因轉錄一條mRNA 27
II. 一個GRS1基因轉譯二個蛋白質 28
III. 粒腺體GlyRS使用non-AUG當轉譯起始密碼 29
IV. GRS1在細胞中的存在位置由訊息胜肽鏈決定 31
V. GRS1利用Leaky scanning作出兩種異構型GlyRS 32
VI. 用西方點漬法證明粒腺體GlyRS使用non-AUG當
轉譯起始密碼 33
VII. 篩選酵母菌中可以使用的各種轉譯起始點 35
VIII. 周圍序列對AUG轉譯效率有明顯差 37
IX. 周圍序列對non-AUG轉譯效率無明顯差異 38
X. 下游二級結構對non-AUG轉譯效率有明顯差異 39
XI. 並列non-AUG（Redundant non-AUG）可增加轉
譯效率 40
第四章討論
I. 粒腺體GlyRS的標的訊號 42
II. GRS1利用leaky scanning轉譯其細胞質及粒腺體
GlyRS 43
III. 下游二級RNA結構對於non-AUG轉譯起始的影
響 45
IV. 罕見的轉譯起始密碼 45
第五章參考文獻 47
圖表 51
附錄一 80
附錄二 81
圖、表目錄
表1、aaRS的分類 51
表2、aaRS的特性 52
表3、周圍序列對ATG轉譯效率的影響 53
表4、周圍序列對non-ATG轉譯效率的影響 54表5、測試並列non-ATG的轉譯機制 55
圖1、aminoacyl-tRNA synthetase（aaRS）的基本生化功能 56
圖2、aaRS在細胞中的功能及角色演化 57
圖3、選擇性轉錄及轉譯機制 58
圖4、sequence context 59
圖5、鑑定GRS1 mRNA的5`端 60
圖6、測試各種不同的GRS1突變基因之互補能力 62
圖7、測試各種不同的GRS1突變基因之互補能力 64
圖8、測試各種不同的VAS1-GRS1融合基因的互補能力 66
圖9、測試各種不同的GRS1-VAS1融合基因的互補能力 68
圖10、在實驗中使用的二級結構序列 70
圖11、利用西方點漬法分析由non-ATG轉譯的蛋白質 71
圖12、GlyRS的兩種異構型蛋白質以leaky scanning的方式
轉譯而來 73
圖13、ALA1的5`端序列 75
圖14、尋找具有轉譯活性的各種可能轉譯起始點 76
圖15、GRS1 non-ATG下游可能存在的二級結構 78
圖16、下游二級結構對non-ATG轉譯效率的影響 79

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指導教授

王健家(Chien-Chia Wang)

審核日期

2003-7-21

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