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| ???org.dspace.app.webui.jsptag.ItemTag.dcfield??? | Value | Language |
|---|---|---|
| dc.contributor.advisor | 溫進德(Jin-Der Wen) | |
| dc.contributor.author | Ren-Hao Liao | en |
| dc.contributor.author | 廖仁豪 | zh_TW |
| dc.date.accessioned | 2022-11-25T03:04:19Z | - |
| dc.date.available | 2026-08-03 | |
| dc.date.copyright | 2021-08-20 | |
| dc.date.issued | 2021 | |
| dc.date.submitted | 2021-08-03 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/81819 | - |
| dc.description.abstract | 細菌中mRNA的轉譯效率顯著受到起始階段的影響。在起始階段，核醣體的30S次單元(亞基)與mRNA的結合，主要是透過位於mRNA起始密碼子上游約6 個鹼基的Shine-Dalgarno (SD) 與30S次單元16S rRNA中的anti-SD (aSD) 序列互補。先前研究表明，SD-aSD 相互作用穩定了30S-mRNA的複合體，並在起始因子 (initiation factor) 和起始tRNA協助下，30S次單元固定在起始密碼子處。然而，關於mRNA 如何招募30S次單元以及30S次單元如何搜索SD序列，得知甚少。在本論文中，我們通過單分子螢光共振能量轉移 (smFRET) 觀察了螢光標記的30S次單元與mRNA的相互作用。我們的觀察表明，30S 亞基首先與位於 5’-非轉譯區 (5’-UTR) 的非特異性位點結合，然後通過未知機制轉移到起始位點。此外，具有長且非結構化 5’-UTR 的人工mRNA (GAA52-sRBS)具有高30S次單元招募效率，但此效率是否與自然發生的mRNA類似則是未知。因此，我們透過和E. coli 中具有高轉譯效率的lpp mRNA進行比較，發現GAA52-sRBS的招募效率為lpp mRNA的11倍。然而，GAA52-sRBS 的實際轉譯效率僅為lpp的50%。此外，透過smFRET的即時觀察，發現30S次單元可能會與lpp 5’-UTR互動。這些結果說明轉譯起始的機制比我們的預期複雜。在後續的實驗中，我們將利用新的螢光標記30S次單元，將標記置於靠近mRNA5’端出口處的S6蛋白，此類30S次單元將可偵測5’-UTR的動態變化，以揭示更多轉譯起始機制的細節。 | zh_TW |
| dc.description.provenance | Made available in DSpace on 2022-11-25T03:04:19Z (GMT). No. of bitstreams: 1 U0001-2907202117574100.pdf: 4730769 bytes, checksum: a536db2552a86621e1127e03feb87ac9 (MD5) Previous issue date: 2021 | en |
| dc.description.tableofcontents | "審定書 i 致謝 ii 摘要 iii Abstract iv Figure list vii Table list viii 1. Introduction 1 1.1 Translation initiation 1 1.2 mRNA recruitment 1 1.3 The hypothetical strategies of 30S subunit searching for RBS 3 1.4 Single-molecule Fluorescence Resonance Energy Transfer (smFRET) 4 1.5 Motivation and purpose 5 2. Materials methods 6 2.1 Material 6 2.1.1 Plasmids 6 2.1.2 Buffer 7 2.1.3 Cell lines 10 2.1.4 Chemicals 10 2.1.5 Kit 12 2.1.6 Enzyme 13 2.1.7 Oligo 13 2.1.8 Constructs 15 2.2 Methods 16 2.2.1 Polymerase Chain Reaction (PCR) for gene cloning 16 2.2.2 Polymerase Chain Reaction (PCR) 17 2.2.3 Plasmid construction and mRNA synthesis 18 2.2.4 Construct 30SS6-ybbR 18 2.2.5 Purification of SFP synthase 22 2.2.6 Labeling reaction of 30SS5-ybbR and 30SS6-ybbR 24 2.2.7 Single-molecule Fluorescence Resonance Energy Transfer assay 25 2.2.8 In vitro translation and Renilla luciferase assay 28 3. Results 29 3.1 Ribosome recruitment of natural mRNA 29 3.1.1 mRNA: GAA52-sRBS may display high recruiting efficiency in the natural environment 29 3.1.2 The length of mRNA may affect the recruiting efficiency in natural mRNA 30 3.1.3 The mRNA with high recruiting efficiency is uncertainly able to enhance translation 30 3.1.4 Real-time observation of Lpp-PC interacting with the 30S by smFRET 31 3.2 New dye-labeled 30S subunits: 30SS6-Cy3 or Cy5 32 3.2.1 S6-ybbR protein expression in E. coli strain JW4158-3 32 3.2.2 The 30SS6-ybbR display normal function in the smFRET system 33 3.2.3 Purification of SFP synthase 33 3.2.4 The 30SS6-ybbR labeled with Cy3-CoA by using SFP synthase 34 3.2.5 smFRET assay of 30SS6-Cy3 34 4. Discussion 36 4.1 Ribosome recruitment of natural mRNA: lpp 36 4.2 New labeled 30S subunit: 30SS6-Cy3 37 4.3 Conclusions 38 Reference: 39 1. Introduction 1 1.1 Translation initiation 1 1.2 mRNA recruitment 1 1.3 The hypothetical strategies of 30S subunit searching for RBS 3 1.4 Single-molecule Fluorescence Resonance Energy Transfer (smFRET) 4 1.5 Motivation and purpose 5 2. Materials methods 6 2.1 Material 6 2.1.1 Plasmids 6 2.1.2 Buffer 7 2.1.3 Cell lines 10 2.1.4 Chemicals 10 2.1.5 Kit 12 2.1.6 Enzyme 13 2.1.7 Oligo 13 2.1.8 Constructs 15 2.2 Methods 16 2.2.1 Polymerase Chain Reaction (PCR) for gene cloning 16 2.2.2 Polymerase Chain Reaction (PCR) 17 2.2.3 Construct plasmid and synthesis mRNA 18 2.2.4 Construct and purify the 30SS6-ybbR 18 2.2.5 Purification of SFP synthase 22 2.2.6 Labeling reaction of 30SS5-ybbR and 30SS6-ybbR 25 2.2.7 Single-molecule Fluorescence Resonance Energy Transfer assay 26 2.2.8 In vitro translation and Renilla luciferase assay 29 3. Results 30 3.1 Ribosome recruitment of natural mRNA 30 3.1.1 mRNA: GAA52-sRBS may display high recruiting efficiency in the natural environment 30 3.1.2 The length of mRNA may affect the recruiting efficiency in natural mRNA 30 3.1.3 The mRNA with high recruiting efficiency is uncertainly able to enhance translation 31 3.1.4 Real-time observation of Lpp-PC interacting with the 30S by smFRET 31 3.2 New dye-labeled 30S subunits: 30SS6-Cy3 or Cy5 33 3.2.1 S6-ybbR protein expression in E. coli strain JW4158-3 33 3.2.2 The 30SS6-ybbR display normal function in the smFRET system 33 3.2.3 Purification of SFP synthase 34 3.2.4 The 30SS6-ybbR labeled with Cy3-CoA by using SFP synthase 34 3.2.5 smFRET assay of 30SS6-Cy3 35 4. Discussion 36 4.1 Ribosome recruitment of natural mRNA: lpp 36 4.2 New labeled 30S subunit: 30SS6-Cy3 37 4.3 Conclusions 38 Reference: 39 Fig. 1 Four hypothetical mechanisms of the translocation of the protein on nucleic acids 45 Fig. 2 The FRET distribution of labeled 30S interaction with mRNA GAA52-sRBS 46 Fig. 3 The location of S6 protein at the 30S and labeling reaction catalyzed by SFP synthase 47 Fig. 4 Plasmid rpsF-ybbR 48 Fig. 5 Construct of Lpp, Lpp-PC (Partial coding region) and Lpp-PC-2 49 Fig. 6 Plasmid: pGAA52-sRBS-Rluc, pGAA6-sRBS-Rluc, pLpp-PC-Rluc and pLpp-5’UTR-Rluc 50 Fig. 7 Flowing cambers assembly and experimental set up of single-molecule FRET 51 Fig. 8 Recruiting efficiency of mRNA: GAA52-sRBS, lpp, and Lpp-PC 52 Fig. 9 Renilla luciferase assay for quantifying translation efficiency 53 Fig. 10 The structure of Lpp-PC-2 and FRET distribution of Lpp-PC-2 interacting with the 30S 54 Fig. 11 Time traces of FRET from Lpp-PC-2 55 Fig. 12 Time traces from Lpp-PC-2+50 nM 30S 56 Fig. 13 IPTG induction test of JW4158-3 expressing S6-ybbR 57 Fig. 14 The FRET distribution of F+18 in the absence and presence of MRE600 30S, 30SS5-ybbR, and 30SS6-ybbR 58 Fig. 15 SDS-PAGE Electrophoresis of SFP synthase 59 Fig. 16 the test of 30SS6-ybbr labeling reaction with Cy3 catalyzed by SFP synthase 60 Fig. 17 Incubation of 30SS6-Cy3 with F+18 mRNA and observation in smFRET system 61 Fig. 18 Real-time observation of 30SS6-Cy3 and F+18 mRNA 62 Table 1 The primers, templates, and primer annealing temperature of PCR 63 Table 2 The restriction enzymes were used for constructing plasmid and linearizing 64 Table 3 Plasmids used or constructed in this thesis 65 " | |
| dc.language.iso | en | |
| dc.subject | 30S次單元(亞基) | zh_TW |
| dc.subject | 轉譯起始 | zh_TW |
| dc.subject | 單分子螢光共振能量轉移 | zh_TW |
| dc.subject | mRNA招募 | zh_TW |
| dc.subject | 核醣體 | zh_TW |
| dc.subject | Translation initiation | en |
| dc.subject | single-molecule Fluorescence Resonance Energy Transfer | en |
| dc.subject | mRNA recruitment | en |
| dc.subject | ribosome | en |
| dc.subject | 30S subunit | en |
| dc.title | 使用單分子螢光共振能量轉移技術研究螢光標記之核醣體亞基如何搜尋訊息核醣核酸的轉譯起始位 | zh_TW |
| dc.title | Using Fluorescence-labeled 30S Ribosomal Subunits to Study How They Find mRNA Translation Initiation Sites by Single-molecule FRET | en |
| dc.date.schoolyear | 109-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 李弘文(Hsin-Tsai Liu),李以仁(Chih-Yang Tseng) | |
| dc.subject.keyword | 轉譯起始,30S次單元(亞基),核醣體,mRNA招募,單分子螢光共振能量轉移, | zh_TW |
| dc.subject.keyword | Translation initiation,30S subunit,ribosome,mRNA recruitment,single-molecule Fluorescence Resonance Energy Transfer, | en |
| dc.relation.page | 65 | |
| dc.identifier.doi | 10.6342/NTU202101906 | |
| dc.rights.note | 同意授權(全球公開) | |
| dc.date.accepted | 2021-08-05 | |
| dc.contributor.author-college | 生命科學院 | zh_TW |
| dc.contributor.author-dept | 分子與細胞生物學研究所 | zh_TW |
| dc.date.embargo-lift | 2026-08-03 | - |
| Appears in Collections: | 分子與細胞生物學研究所 | |
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| U0001-2907202117574100.pdf Until 2026-08-03 | 4.62 MB | Adobe PDF |
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