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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 袁小琀 | |
dc.contributor.author | Wei-Hsuan Tang | en |
dc.contributor.author | 唐偉軒 | zh_TW |
dc.date.accessioned | 2021-06-17T08:27:57Z | - |
dc.date.available | 2019-08-27 | |
dc.date.copyright | 2019-08-27 | |
dc.date.issued | 2019 | |
dc.date.submitted | 2019-08-12 | |
dc.identifier.citation | 五、參考文獻
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/74285 | - |
dc.description.abstract | 粒線體在細胞中的主要功能是產生能量供細胞使用,在真核細胞中是不可或缺的胞器。而粒線體的缺失亦會導致數種人類疾病。為了維持粒線體功能的正常,RNA的降解在粒線體基因表現以及RNA品質控管上扮演重要角色。人類粒線體中,二聚體的解旋酶Suv3與三聚體的外切酶PNPase形成五聚體的RNA降解體,將具有二級結構的RNA解開,並從3'端往5'端分解。雖然單體的Suv3結構已經被報導,但Suv3是如何形成二聚體,並與PNPase交互作用形成RNA降解體,來合作解開並分解RNA仍然未知。
為了瞭解Suv3如何解開雙股RNA,我們從大腸桿菌表達了刪除N端粒線體位置序列(mitochondrial localization sequence)的人類粒線體解旋酶Suv3(Suv3)及C端刪除蛋白(Suv3ΔC)。純化後的Suv3與Suv3ΔC分別以二聚體與單體存在,說明Suv3的C端與形成二聚體有關。我們發現二聚體Suv3相較於單體Suv3ΔC對單股RNA的結合親和力強10倍,並且二聚體Suv3不論ATP或ADP是否存在下,仍可維持與單股RNA結合的能力。而單體Suv3ΔC只有在ATP存在時有較高的單股RNA結合親和力。解旋酶活性測試中,當有ATP時,Suv3可解開帶有3' overhang的雙股RNA,而二聚體Suv3相較於單體Suv3ΔC的解旋酶活性略好。Suv3的二聚體結構不僅促進解旋酶活性,更重要的是與PNPase交互作用。綜合以上實驗結果,Suv3與RNA受質的結合不受ATP或ADP調控,說明在ATP水解後,Suv3會持續與RNA結合並繼續解開雙股RNA,被解開的單股RNA則被與Suv3交互作用的PNPase連續的降解掉。最後總結,二聚體Suv3與三聚體PNPase一起形成一個有效率的粒線體RNA降解體,可以持續的解開並分解粒線體中具有二級結構的RNA。 | zh_TW |
dc.description.abstract | Mitochondria are indispensable organelles in all eukaryotes as they provide cellular energy in the form of ATP. Defects in mitochondrial functions thus lead to various mitochondria disorder and human diseases. To maintain the normal function of mitochondria, RNA turnover plays an important part in the regulation of mitochondrial gene expression and the surveillance of abnormal RNA molecules. In human mitochondria, dimeric helicase Suv3 and trimeric polynucleotide phosphorylase (PNPase) form a pentameic RNA exosome for unwinding and degrading structured RNA from 3' to 5' ends. Although the structure of monomeric Suv3 was reported, it remains unclear how Suv3 is assembled into a homodimer to interact with PNPase in the mitochondrial RNA exosome for corporative RNA unwinding and degradation.
To understand how Suv3 unwinds a RNA duplex, we expressed human Suv3 (residues 44-786) and a C-terminal tail-truncated Suv3, named Suv3ΔC (residues 44-722) in E. coli, both without the N-terminal mitochondrial localization sequence (MLS). The purified recombinant Suv3 and Suv3ΔC formed dimers and monomers, respectively, suggesting that the C-terminal tail of Suv3 mediates protein dimerization. We found that Suv3 had a 10-fold greater RNA-binding affinity compared with Suv3ΔC at pH7.4. Suv3 bound single-stranded RNA with high affinities in the presence or absence of ATP and ADP, while Suv3ΔC bound RNA with a high affinity only in the presence of ATP. Suv3 had a slightly higher ATP-dependent helicase activity than Suv3ΔC in unwinding a duplex RNA with a 3'-overhang. Dimeric conformation of Suv3 is important not only for promoting the ATP-dependent RNA helicase activity but also for interaction with PNPase. Taken together these results suggest that RNA is continuously bound with Suv3 after ATP hydrolysis and RNA unwinding, and the unwound single-stranded RNA is degraded processively by the Suv3-interacting PNPase. We thus conclude that dimeric Suv3 and trimeric PNPase form a robust machinery of mitochondrial RNA exosome for efficient processive RNA unwinding and degradation. | en |
dc.description.provenance | Made available in DSpace on 2021-06-17T08:27:57Z (GMT). No. of bitstreams: 1 ntu-108-R06442020-1.pdf: 4278273 bytes, checksum: 8e558e3b2d8232a39c5ae33137f6324d (MD5) Previous issue date: 2019 | en |
dc.description.tableofcontents | 中文摘要 1
ABSTRACT 2 一、介紹 1.1 背景 3 1.2 真核生物細胞質的mRNA降解路徑與RNA降解體 3 1.3 酵母菌粒線體的RNA降解體 4 1.4 人類粒線體的RNA降解體 5 1.5 Suv3的結構與功能 6 1.6 PNPase的結構與功能 7 1.7 研究目的 8 二、材料與方法 2.1 蛋白質表現質體建構 9 2.2 重組蛋白質的表現與純化 9 2.3 蛋白質電泳 10 2.4 小角繞射 (Small-angle X-ray scattering) 10 2.5 偏極化螢光試驗 (Fluorescence polarization assay) 10 2.6 解旋酶活性試驗 (Helicase activity assay) 11 2.7 蛋白質養晶 11 三、實驗結果 3.1 缺失C端區域的Suv3 (Suv3ΔC)無法形成二聚體 13 3.2 二聚體Suv3與三聚體PNPase形成穩定五聚體,但Suv3ΔC無法與PNPase形成穩定五聚體 13 3.3 Suv3與PNPase形成五聚體之小角散射結構 14 3.4 二聚體Suv3與單股RNA結合親和力比單體Suv3ΔC較高 15 3.5 二聚體Suv3與單股RNA結合的親和力較不受ATP或ADP調控 15 3.6 二聚體Suv3的解旋活性比單體Suv3ΔC較佳 16 3.7 PNPase與二聚體Suv3的交互作用促進Suv3的解旋酶活性 16 3.8 Suv3_V65E/F150E/T282E/Y334E為二聚體具解旋酶活性且可溶性較Suv3佳 17 3.9 Suv3_V65E/F150E/T282E/Y334E的結晶 18 四、討論 4.1 Suv3解旋機制探討 19 4.2 Suv3 C端區域的序列分析與結構預測 20 五、參考文獻 21 六、圖表 圖一、真核生物細胞質中的mRNA降解路徑 26 圖二、真核生物細胞核與細胞質的RNA降解體(exosome) 27 圖三、酵母菌粒線體RNA降解體的晶體結構 28 圖四、人類粒線體核酸酶 29 圖五、Suv3與其他SF2解旋酶序列比較 30 圖六、Suv3單體的晶體結構 31 圖七、人類PNPase的晶體結構 32 圖八、Suv3重組蛋白純化 33 圖九、Suv3ΔC重組蛋白純化 34 圖十、Suv3為二聚體,但Suv3ΔC為單體 35 圖十一、二聚體Suv3與三聚體PNPase形成五聚體,但單體Suv3ΔC無法與三聚體PNPase形成五聚體 36 圖十二、Suv3、PNPase以及PNPase-Suv3複合體之小角散射分子構型 37 圖十三、Suv3與Suv3ΔC對單股RNA的結合親和力之測定 38 圖十四、Suv3與Suv3ΔC在有不同ATP相似物時對單股RNA的結合親和力之測定 39 圖十五、Suv3與Suv3ΔC的解旋酶活性 40 圖十六、PNPase促進二聚體Suv3的解旋酶活性之分析 41 圖十七、PNPase無法促進單體Suv3ΔC的解旋酶活性 42 圖十八、增加Suv3可溶性的突變 43 圖十九、Suv3_V65E/F150E/T282E/Y334E重組蛋白純化及解旋酶活性測試 44 圖二十、Suv3_V65E/F150E/T282E/Y334E晶體 46 圖二十一、人類與酵母菌Suv3胺基酸序列 47 圖二十二、Suv3 C端區域胺基酸序列分析及二級結構預測 48 | |
dc.language.iso | zh-TW | |
dc.title | 人類粒線體解旋酶Suv3的生化特性 | zh_TW |
dc.title | Biochemical characterization of the human mitochondria helicase Suv3 | en |
dc.type | Thesis | |
dc.date.schoolyear | 107-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 詹迺立,曾秀如 | |
dc.subject.keyword | 解旋?,RNA降解,粒線體RNA剪接, | zh_TW |
dc.subject.keyword | helicase, RNA turnover,RNA decay,mitochondrial RNA processing, | en |
dc.relation.page | 49 | |
dc.identifier.doi | 10.6342/NTU201903334 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2019-08-13 | |
dc.contributor.author-college | 醫學院 | zh_TW |
dc.contributor.author-dept | 生物化學暨分子生物學研究所 | zh_TW |
顯示於系所單位: | 生物化學暨分子生物學科研究所 |
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