探討酵母菌GlyRS基因的表現及功能

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陳順佳(Shun-Jia Chen) 查詢紙本館藏

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論文名稱

探討酵母菌GlyRS基因的表現及功能
(Study of the expression and function of yeast GlyRS genes)

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摘要(中)

之前的研究發現釀酒酵母中ALA1及GRS1基因都是使用non-AUG起始密碼轉譯出各自的粒線體型蛋白質。我們實驗室研究發現non-AUG轉譯起始效率大約是AUG的三分之一或更少，而且周邊序列對non-AUG的轉譯效率影響也較對AUG大，除此之外，也發現最好的周邊序列是AAA (-3到-1核甘酸)。重複的non-AUG起始密碼(如ACGACG)通常能增加轉譯的效率；但某些重複的non-AUG起始密碼(如GUGGUG)會使周邊序列變差(尤其是-3核甘酸)，則其轉譯效率反而變差。另一個重大發現是：絕大部分酵母菌都只含有一個glycyl-tRNA synthetase (GlyRS)基因(稱做GRS1)，但是S. cerevisiae及V. polyspora卻有兩個相異的GlyRS基因(稱做GRS1及GRS2)。研究結果顯示：所有的酵母菌GRS1基因都是雙功能的，能同時做出細胞質及粒線體的GlyRS異構型，但是GRS2基因卻是非必需的。然而最令人驚奇的是：GRS2基因雖然在正常培養條件下不表現，它卻可以被一些逆境條件誘導表現，例如鹼性培養基(pH 8.0)或高溫生長條件(37°C)，且純化出來的GlyRS2蛋白質具有相當程度的胺醯化活性。在正常(30°C)及高溫條件下(37°C) GlyRS1與GlyRS2的穩定度都相當高，且在特定高溫條件下GRS2可以互補GRS1的剔除株，維持其正常生長。也許被誘導出來的GlyRS2在某些條件下可以取代GlyRS1的功能，另一種可能性則是GlyRS2參與其它代謝機制。

摘要(英)

Previous studies showed that ALA1 and GRS1 of Saccharomyces cerevisiae can initiate translation of their respective mitochondrial forms from a non-AUG codon. Our results showed that the translation efficiency of non-AUG initiation is about 30% (or less) relative to that of AUG initiation. In addition, it appeared that a non-AUG initiator codon is much more sensitive to its sequence context than is an AUG initiator codon, and AAA (the nucleotides at position -3 to -1 relative to the initiator) is the most favorable sequence context. Moreover, redundancy of non-AUG initiators, for instance ACGACG, significantly increased the translational efficiency. However, some redundant non-AUG initiators such as UUGUUG that have a poor sequence context (especially at position -3 relative to the second UUG codon), reduced the efficiency of translation. Another interesting discovery reported here was that the majority of yeast species possess a single glycyl-tRNA synthetase (GlyRS) gene (named GRS1). In contrast, S. cerevisiae and Vanderwaltozyma polyspora possessed two GlyRS genes (named GRS1 and GRS2). In all cases, GRS1 was dual-functional, because it encodes both cytoplasmic and mitochondrial forms of GlyRS. In contrast, GRS2 was pseudogene-like and dispensable for growth. Surprisingly, while GRS2 was silent under normal growth conditions (30°C), its expression was induced by certain stresses such as high temperature (37°C) and high external pH (pH 8.0). In addition, purified recombinant GlyRS2 retained a substantial level of aminoacylation activity. Both GlyRS1 and GlyRS2 were appreciably stable in vivo. When overexpressed, the GRS2 gene could rescue the growth defect of a GRS1 knockout strain. Altogether, these data suggest that GRS2 may function to substitute for GRS1 under certain circumstances. Alternatively, it may be involved in other as-yet-unidentified metabolic pathways.

關鍵字(中)

關鍵字(英)

★ aminoacyl-tRNA synthetase
★ inducible gene
★ sequence context
★ aminoacylation

論文目次

Table of contents
中文摘要 i
Abstract ii
誌謝辭 iii
Table of contents iV
List of Figures Vi
Overall introduction 1
Chapter I Translational efficiency of a non-AUG initiation codon is significantly affected by its sequence context in yeast 4
Abstract 5
Introduction 6
Experimental procedures 9
Results 12
Discussion 18
Chapter II Translational efficiency of redundant ACG initiator codons is enhanced by a favorable sequence context and remedial initiation 23
Abstract 24
Introduction 25
Experimental procedures 28
Results 32
Disscusion 40
Chapter III Vanderwaltozyma polyspora possesses a housekeeping and an inducible glycyl-tRNA synthetase gene 45
Abstract 46
Introduction 47
Experimental procedures 48
Results 52
Discussion 59
Summary 63
Reference 64
Figures 70
List of Figures
Figure 1. The 5’-end sequence of GRS1. 70
Figure 2. Assay of protein expression patterns by Western blots. 71
Figure 3. Screening for important flanking sequences. 73
Figure 4. Effect of sequence context on the native TTG initiator. 75
Figure 5. Effect of sequence context on ATG and non-ATG initiators. 78
Figure 6. Context effect determined using lacZ as a reporter. 79
Figure 7. Efficiencies of translation initiation from various redundant and single non-AUG initiation codons. 82
Figure 8. Effect of the penultimate amino-terminal residue on the turnover of AlaRS-LexA fusions. 85
Figure 9. Efficiencies of translation initiation from various combinations of non-AUG initiation codons. 86
Figure 10. Effect of the nucleotide at position -3 on non-AUG initiation. 88
Figure 11. Efficiency of translation initiation from three successive ACG codons. 89
Figure 12. Efficiency of translation initiation from non-successive ACG codons. 91
Figure 13. Functional substitution of ATG1 of VAS1 with redundant GTG codons. 92
Figure 14. Comparison of yeast GlyRS1 and GlyRS2. 94
Figure 15. Cross-species complementation assays for yeast GlyRS genes. 95
Figure 16. Complementation assays for yeast GRS2 genes cloned into pADH. 97
Figure 17. Mapping the translation initiator codons of various yeast GlyRS genes. 100
Figure 18. Relative mRNA expression of GRS1 and GRS2 in Vanderwaltozyma polyspora and Saccharomyces cerevisiae. 102
Figure 19. Complementation assays for yeast GRS1 and GRS2 genes. 104
Figure 20. Protein stability assay for GlyRSs in vivo. 106
Figure 21. Aminoacylation assay for ScGlyRS1 and ScGlyRS2. 107
Figure 22. Aminoacylation assay for VpGlyRS1 and VpGlyRS2. 108
Figure 23. Aminoacylation assay at different temperatures. 109
Figure 24. Phylogenetic analysis of α2-dimeric GlyRS proteins. 111

參考文獻

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指導教授

王健家(Chien-Chia Wang)

審核日期

2011-1-24

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