博碩士論文 107821604 詳細資訊

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姓名 唐山姆(Samuel Halomoan Tambunan)  查詢紙本館藏   畢業系所 生命科學系
(Recognition of tRNA His isoacceptors by human HisRS isoforms)
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摘要(中) 人類細胞核基因體擁有兩個不同的基因,分別解碼細胞質及粒線體histidyl-tRNA synthetase (HisRS),細胞質中的HisRS ( HsHisRSc)主要辨識G-1和A73核苷酸;相反的,粒線體內的HisRS ( HsHisRSm)不僅辨識G-1:C73,也辨識粒線體tRNAHis的反密碼。在此篇論文中,我們探討人類野生型與突變型HisRS對於tRNAHis G-1和G34 (反密碼子上最有影響力的核苷酸)的辨識,胺基酸R169中負責辨識G-1, 胺基酸EQE (469 to 471)負責辨識G34,隨後我們將此進行突變,分析其互補活性 (體內)與胺醯化活性(體外),有趣的是,突變EQE時,削弱了部分HsHisRSc的活性,而 R169 的突變則完全阻斷了其體外和體內活性。相比之下,HsHisRSm 的 EQE 或 R170(相當於HsHisRSc 中的 R169)突變完全阻斷了體內活性。此外,HsHisRSc 會結合自身的 mRNA 的一個小片段,這個小片段會摺疊成反密碼子類似結構(一種具有 GUG 的反密碼子的stem-loop結構),這個結果顯示HsHisRSc可能可以抑制它自己的蛋白質表達。本研究強調二個演化相近的HisRS異構酶具有截然不同的tRNA喜好。
摘要(英) Humans possess two distinct nuclear genes encoding closely related mitochondrial and cytoplasmic isoforms of histidyl-tRNA synthetase (HisRS), respectively. The human cytoplasmic form of HisRS (HsHisRSc) recognizes a G nucleotide with 5’-monophosphate at position -1 (G-1) and A73 as the major identity elements. In contrast, its mitochondrial counterpart (HsHisRSm) recognizes not only G-1:C73 but also the anticodon of the mitochondrial tRNAHis. In this thesis, we focused on recognition of G-1 and G34 (the most influential nucleotide of the anticodon) of human tRNAHis isoacceptors by the WT and mutant HisRS isoforms. The amino acid R169 in the motif 2, which is responsible for recognition of G-1, and EQE (469 to 471) in the anticodon-binding domain, which is responsible for recognition of G34, were mutated, and the complementation (in vivo) and aminoacylation activities (in vitro) of the resultant mutants were analyzed. Interestingly, mutation at EQE partially impaired the activity of HsHisRSc, while mutation at R169 completely blocked its activity in vitro and in vivo. In contrast, mutation at EQE or R170 of HsHisRSm (equivalent to R169 of HsHisRSc) completely blocked its activity in vivo. In addition, HsHisRSc robustly bound an anticodon mimic (an anticodon-like stem-loop structure with GUG in the loop) retrieved from its own mRNA, suggesting that this enzyme might regulate its own expression via autogenous translation repression. This study underscores the specialized tRNA preferences of two closely related HisRS isoforms.
關鍵字(中) ★ 氨酰-tRNA 合成酶
★ 遗传密码
★ 蛋白质合成
★ 翻译
★ tRNA
關鍵字(英) ★ aminoacyl-tRNA synthetase
★ genetic code
★ protein synthesis
★ translation
★ tRNA
ABSTRACT (In Chinese) ii
1.1 Protein biosynthesis 1
1.2 Aminoacyl-tRNA synthetases 1
1.3 Histidyl-tRNA synthetase 2
1.4 The canonical identity element G-1 of tRNAHis and its recognition by HisRS 3
1.5 Homo sapiens HisRS and tRNAHis 4
2.1 Cloning and purification of human HisRS variants 6
2.2 Complementation assays for cytoplasmic and mitochondrial activities 7
2.3 In vitro transcription of tRNAHis variants and an anticodon mimic 8
2.4 Aminoacylation assay 8
2.5 Electrophoretic mobility shift assay 9
3.1 HsHisRS isoforms recognize divergent discriminator bases (A73 or C73) 10
3.2 Mutation at “EQE” impaired the rescue activity of both HsHisRS isoforms 11
3.3 Mutation at “EQE” reduced the aminoacylation activity of HsHisRSm 12
3.4 Overexpression of SctRNAnHis improved the rescue activity of the “EQE” mutant of HsHisRSc 13
3.5 Mutation at R169/R170 blocked the catalytic activity of both HsHisRS isoforms 14
3.6 HsHisRSc bound to a putative anticodon mimic retrieved from its mRNA 15
4.1 HsHisRSc recognizes the anticodon in tRNAnHis as the minor identity element 17
4.2 HsHisRSm specifies the anticodon in tRNAmHis as the one of the major identity elements 18
4.3 HsHisRSc and HsHisRSm identify 5’monophosphorylated G-1 in tRNAmHis as the major identity element 18
4.4 Human HisRS binds to its own mRNA 20
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指導教授 王健家(Chien-Chia Wang) 審核日期 2021-7-19
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