Functional Repurposing of C-Ala Domains

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娜其菈(KUN ROHMATAN NAZILAH) 查詢紙本館藏

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論文名稱

(Functional Repurposing of C-Ala Domains)

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★ tRNA aminoacylation by a naturally occurring mini-AlaRS	★ Recognition of a non-canonical tRNAAla by a non-canonical alanyl-tRNA synthetase
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摘要(中)

alanyl-tRNA synthetase（丙胺酸-tRNA合成酶）是至今唯一仍保留其
原型結構的胺基酸-tRNA合成酶 (aminoacyl-tRNA synthetase)。AlaRS 原型結
構由催化、tRNA辨認、編輯和 C-Ala 結構區域共同組成。在 AlaRS 的四個
結構區域中，C-Ala 的蛋白質序列變異最大，因此在演化過程中，伴隨著功
能上的轉變。大腸桿菌C-Ala 強力結合 tRNA ，且在胺醯化扮演重要功能，
人類C-Ala 強力結合DNA，但是在胺醯化反應中卻是可有可無。為了進一步
了解C-Ala的功能及演化，我們研究及分析二個演化上疏離的低等真核生物
C-Ala的核酸結合能力。我們發現酵母菌Saccharomyces cerevisiae的 C-Ala與
tRNAAla 有很強的結合能力，但是不與 DNA 結合，在胺醯化反應中扮演重要
角色，這個特性與大腸桿菌 C-Ala 相似；然而黏菌Dictyostelium discoideum
C-Ala 則可以同時結合tRNAAla與DNA，且在胺醯化反應中扮演重要功能，
這個特性與線蟲 C-Ala 相似。這些結果顯示，C-Ala由原核演化到真核過程
中，它的功能也由tRNA結合逐漸演變成DNA結合，而一些真核細胞的C-Ala
可能可以同時結合tRNAAla及DNA

摘要(英)

AlaRS is the only aminoacyl-tRNA synthetase (aaRS) that still retains a
conserved prototype structure. AlaRS consists of catalytic, tRNA-recognition,
editing, and C-Ala domains. Among these four domains, C-Ala is highly diverged
in sequence. E. coli C-Ala robustly binds tRNA and plays an important role in
dimerization and aminoacylation, while human C-Ala robustly binds DNA and is
dispensable for aminoacylation. Paradoxically, C. elegans (nematode) C-Ala
robustly binds both tRNA and DNA and plays an important role in aminoacylation.
To gain further insight into the evolution of C-Ala, we explored the nucleic acidbinding properties of C-Ala domains obtained from distantly-related lower
eukaryotes. Our data showed that Saccharomyces cerevisiae C-Ala binds tRNAAla
but not DNA and plays an important role in aminoacylation, a feature similar to E.
coli C-Ala, whereas Dictyostelium discoideum (slime mold) C-Ala binds both
tRNAAla and DNA and plays an important role in aminoacylation, a feature similar
to C. elegans C-Ala. It thus appears that as prokaryotes evolved to eukaryotes, CAla has been repurposed from mediating tRNAAla binding to DNA binding, with
certain eukaryotes binding to both ligands.

關鍵字(中)

★ alanyl-tRNA synthetase
★ C-Ala
★ 演化
★ 蛋白質合成
★ 轉譯

關鍵字(英)

★ alanyl-tRNA synthetase
★ C-Ala
★ evolution
★ protein synthesis
★ translation

論文目次

TABLE OF CONTENT
ABSTRACT (in Chinese) i
ABSTRACT ii
ACKNOWLEDGEMENT iii
TABLE OF CONTENT iv
LIST OF FIGURES vi
ABBREVIATION vii
CHAPTER I INTRODUCTION 1
1.1 Aminoacyl tRNA synthetases 1
1.2 Alanyl-tRNA synthetase 2
1.3 C-Ala functions in several organisms 3
1.4 Specific aim 5
CHAPTER II MATERIALS and METHODS 6
2.1 Plasmid construction 6
2.2 Purification of full length of AlaRS, AlaRS (ΔC-Ala), and C-Ala domain of
slime mold 7
2.3 In vitro transcription of tRNAAla 8
2.4 5’ -end labeling of tRNA with γ-32P ATP 8
2.5 Nucleic binding assay 9
2.6 Aminoacylation 9
2.7 Phylogenetic Analyses 9
CHAPTER III RESULT 11
3.1 Phylogenetic tree of C-Ala domain 11
3.2 Yeast C-Ala strongly binds to tRNA but poorly binds to DNA 11
3.3 Slime mold AlaRSc (ΔC-Ala) was successfully purified with the purity level
similar to the wild type enzyme 12
3.4 Slime mold AlaRSc (ΔC-Ala) failed to charge DdtRNAnAla 13
3.5 Slime mold C-Ala protein was successfully purified to the high purity 13
3.6 Slime mold C-Ala strongly binds to tRNA and modestly binds to DNA 14
CHAPTER IV DISCUSSION 16
4.1 Lower eukaryotic C-Ala behaves like prokaryotic C-Ala 16
4.2 In vivo isolated tRNA is better substrate for aminoacylation 18
4.3 Yeast and slime mold AlaRSs possess non-canonical function in DNA
binding domain 18
4.4 Evolutionary relationship of C-Ala 19
LIST OF FIGURES 21
REFERENCES 32
APPENDIX A 34
PRIMER LIST 34
APPENDIX B 35
PLASMID LIST 35

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指導教授

王健家(Chien-Chia Wang)

審核日期

2022-7-19

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