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研究生:廖仁豪
研究生(外文):Ren-Hao Liao
論文名稱: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
論文名稱(外文):Using Fluorescence-labeled 30S Ribosomal Subunits to Study How They Find mRNA Translation Initiation Sites by Single-molecule FRET
指導教授:溫進德
指導教授(外文):Jin-Der Wen
口試委員:李弘文李以仁
口試委員(外文):Hung-Wen LiI-Ren Lee
口試日期:2021-07-07
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:分子與細胞生物學研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2021
畢業學年度:109
語文別:英文
論文頁數:65
中文關鍵詞: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
外文關鍵詞:Translation initiation30S subunitribosomemRNA recruitmentsingle-molecule Fluorescence Resonance Energy Transfer
DOI:10.6342/NTU202101906
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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
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