博碩士論文 105821603 詳細資訊

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姓名 尼古拉(Restu Nugraha)  查詢紙本館藏   畢業系所 生命科學系
論文名稱 Effect of Biotinylation on the Structure and Function of a Yeast tRNA-Binding Protein
(Effect of Biotinylation on the Structure and Function of a Yeast tRNA-Binding Protein)
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摘要(中) 中文摘要

Arc1p是一個酵母菌tRNA結合蛋白質,它能與細胞質中的 glutamyl-tRNA synthetase(GluRSc)和 methionyl-tRNA Synthetase(MetRS)形成三元複合物。形成複合物可以顯著提高這兩種酵素對其相對應tRNA的胺醯化效率。最近的研究發現,Arc1p 的 N 端區域Lys86可以被轉譯後修飾,加上一個生物素(biotin)為了進一步研究生物素化(biotinylation)對Arc1p的結構及功能影響,我們進一步純化及比較野生型及突變型Arc1p的結構差異,結果發現發現:K86R和K86A突變完全阻斷了Arc1p生物素化,但是不會影響該蛋白質的細胞內功能,突變Arc1p仍能解救ARC1剔除株的cold sensitive外表型。然而,生物素化將會將影響Arc1p的熱穩定性,且 K86R在蛋白酶處理下看起來更耐受,而K86A更敏感。

摘要(英) Abstract

Arc1p is a yeast-specific tRNA-binding protein that has ability to form a ternary complex with glutamyl-tRNA synthetase (GluRSc) and methionyl-tRNA synthetase (MetRS) in the cytoplasm. This complex can significantly enhance the aminoacylation efficiency of these two aaRSs to their respective cognate tRNAs. Recently, it was found that Arc1p can be biotinylated via post-translational modification at Lys86 (K86) in the N-domain. We herein studied the effect of K86 mutation on Arc1p’s structure and function. We found that mutation in K86R and K86A dramatically decreased the biotinylation level of Arc1p and altered its secondary structure. However, the mutant Arc1p could effectively rescue the cold-sensitive phenotype of an ARC1- strain, suggesting that biotinylation is dispensable for the rescue activity of Arc1p. Interestingly, K86R was more resistant to protease treatment than the wild-type, while K86A was more sensitive.

Keywords: Arc1p, biotinylation, K86A, K86R, mutation.
關鍵字(中) ★ 生物素化
★ 突變
關鍵字(英) ★ Biotinylation
★ Arc1p
論文目次 Table of Content

Abstract i
Acknowledgments ii
Table of content iii
List of Figure iv
Abbreviation v
Chapter I – Introduction 1
Background 1
Objectives 2
Outlines 2
Chapter II – Literature Review 3
2. 1 Arc1p and Its Properties 3
2. 2 Role and Function of Arc1p 4
2. 3 Aminoacyl-tRNA Synthetase (aaRS) 4
2. 4 Multi-aminoacyl-tRNA Synthetase 6
2. 5 Biotinylation 7
Chapter III – Materials and Methods 9
3. 1 Strain, Culture Medium, and Transformation 9
3. 2 Plasmid Construction 11
3. 3 Arc1p Purification 11
3. 4 Western Blotting Method 13
3. 5 Streptavidin-based Gel Mobility Shift Assay 14
3. 6 Circular Dichroism (CD) Spectroscopy 15
3. 7 Limited Proteolysis 15
Chapter IV – Result 16
4.1 Biotinylation of Yeast Arc1p and Variants 16
4.2 Biotinylation of Arc1p’s is not Required for the Rescue Activity 16
4.3 Effect of Biotinylation on the Secondary Structure in Vitro of Arc1p 17
4.4 Purification and Characterization of Arc1p Variant 17

Chapter V – Discussion and Conclusion 19
5.1 Discussion 19
5.2 Conclusion 20
Reference 21
Appendix 25
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指導教授 王健家 Widodo(Chien-Chia Wang Widodo) 審核日期 2018-5-2
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