秀麗隱桿線蟲線粒體AlaRS通過非傳統模式識別T型無臂tRNAAla

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瑪希塔(Masita Imamsari) 查詢紙本館藏

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生命科學系

論文名稱

秀麗隱桿線蟲線粒體AlaRS通過非傳統模式識別T型無臂tRNAAla
(Caenorhabditis elegans mitochondrial AlaRS recognizes a T-armless tRNAAla through a non-traditional mode)

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摘要(中)

Aminoacyl-tRNA synthetases（aaRSs）是一群普遍存在的酵素，它們的主要功能是將特定胺基酸接到相對應的tRNA，例如alanyl-tRNA synthetase（AlaRS）會將Ala接到tRNAAla 。 AlaRS具有四個重要的結構區，來維持完整的功能，包括催化區、tRNA結合區、編輯區、C-Ala區。這四個結構區是高度保留的，並且提供了完整的tRNA胺醯化及編輯能力。有趣的是，秀麗隱桿線蟲的粒線體AlaRS（CeAlaRSm）缺少C-Ala區，且其對應的tRNAAla缺乏TΨC，即便如此，CeAlaRSm仍然能精確地辨認tRNAAla上的辨識元素G3:U70配對。然而，我們的實驗結果發現CeAlaRSm只能有效胺醯化微型tRNAAla (microhelix-Ala)，卻無法有效胺醯化完整的tRNAAla，如果將線蟲細胞質AlaRS（CeAlaRSc）的C-Ala區與CeAlaRSm融合產生融合酶，這個融合酶在in vivo和in vitro的情形下皆能更有效地胺醯化tRNAAla。然而，從CeAlaRSc中移除C-Ala區後，其胺醯化功能將產生缺陷。另外，一般AlaRS的tRNA結合區都含有兩個高度保留的胺基酸Asn和Asp，這二個胺基酸主要用來辨識tRNAAla 中的辨識元素(identity element) G3：U70。然而，CeAlaRSm在相對應位置的胺基酸卻是Gly和Glu。若將Gly322或Glu420突變為Ala，將導致酵素的胺醯化活性降低(in vitro)，以及失去提供酵母菌AlaRS剔除株存活所需的胺醯化能力(in vivo)，這結果表示，雖然CeAlaRSm沒有高度保留的胺基酸Asn及Asp，Gly及Glu仍然參與tRNAAla的G3:U70辨認。我們的結果顯示，CeAlaRSm是一種非典型的AlaRS，可通過非傳統的模式來辨認缺乏TΨC的 tRNAAla。

摘要(英)

Aminoacyl-tRNA synthetases (aaRSs) are a family of ubiquitously expressed enzymes, attaching a specific amino acid to its corresponding tRNA, such as alanyl-tRNA synthetase (AlaRS) attaching alanine to tRNAAla. AlaRS consists of four important domains to work properly, including catalytic domain, tRNA-binding domain, editing domain, and C-Ala domain. Those four domains are highly conserved and support the full function of tRNA alanylation. Interestingly, C. elegans mitochondrial AlaRS (CeAlaRSm) lacks C-Ala domain. Our results showed that CeAlaRSm retains its tRNA specificity, but poorly recognizes a full-length tRNAAla in vivo and in vitro. In addition, we showed that fusion of the C-Ala domain of C. elegans cytoplasmic AlaRS (CeAlaRSc) to CeAlaRSm results in a fusion enzyme that can more efficienty charge tRNAAla in vivo and in vitro. On the other hand, deletion of the C-Ala domain from CeAlaRSc yielded a truncated enzyme defective in aminoacylation. Unlike canonical AlaRSs, which contain two highly conserved amino acid residues, Asn and Asp for recognition of the canonical identity element G3:U70 in the tRNAAla. CeAlaRSm contains Gly and Glu which at the corresponding positions. Mutation of G322 to A or E420 to A led to an enzyme defective in aminoacylation in vitro and complementation in vivo. Our results suggest that CeAlaRSm is a non-canonical AlaRS that recognizes a T-armless tRNAAla through a non-traditional mode.

關鍵字(中)

★ AlaRS
★ tRNA胺醯化
★ C-Ala結構區
★ 辨識元素(identity element)
★ tRNAAla

關鍵字(英)

★ AlaRS
★ aminoacylation
★ C-Ala domain
★ identity element
★ tRNAAla

論文目次

ABSTRACT i
ABSTRACT (In Chinese) ii
ACKNOWLEDGEMENT iii
TABLE OF CONTENT iv
CHAPTER I INTRODUCTION 1
I. Aminoacyl-tRNA Synthetases (aaRSs) are group of translation enzymes 1
II. Alanyl-tRNA Synthetase (AlaRS) is a class II aaRS 1
III. AlaRS identifies tRNAAla through G3:U70 2
IV. Non-canonical AlaRSs and tRNAsAla 3
V. C. elegans AlaRS and tRNAAla 4
CHAPTER II MATERIALS AND METHODS 6
I. Construction and purification of CeAlaRSm 6
II. Heterologous complementation assay 7
III. Western blotting 8
IV. Construction and in vitro transcription of tRNAsAla 8
V. Aminoacylation assay 9
CHAPTER III RESULTS 11
I. Rescue of yeast AlaRS knockout strain by CeAlaRSm 11
II. CeAlaRSm cannot efficiently charge SctRNAnAla and EctRNAAla in vitro 12
III. The C-Ala domain increases the aminoacylation activity of CeAlaRSm towards full-length tRNAAla 13
IV. Mutation of G322 or E420 to A impairs the aminoacylation activity of CeAlaRSm in vivo and in vitro 14
V. Overexpression of tRNAAla rescues the defective phenotype of CeAlaRSm mutants in vivo 15
CHAPTER IV DISCUSSIONS 17
I. CeAlaRSm poorly charges full-length tRNAAla due to lack of the C-Ala domain 17
II. CeAlaRSm lacks the highly conserved Asn and Asp for G3:U70 recognition 18
III. Phylogenetic analysis of CeAlaRSc and CeAlaRSm 19
REFERENCES 21
APPENDIX A 46
APPENDIX B 50
APPENDIX C 53

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指導教授

王健家(Chien-Chia Wang)

審核日期

2020-12-16

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