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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
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dc.contributor.advisor | 溫進德(Jin-Der Wen) | |
dc.contributor.author | Cheng-Han Yang | en |
dc.contributor.author | 楊承翰 | zh_TW |
dc.date.accessioned | 2021-06-17T08:11:19Z | - |
dc.date.available | 2024-08-22 | |
dc.date.copyright | 2019-08-22 | |
dc.date.issued | 2019 | |
dc.date.submitted | 2019-08-15 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/73830 | - |
dc.description.abstract | 轉譯作用的起始階段在於調控蛋白質生成中扮演著舉足輕重的地位,作為一個速率決定步驟,它掌控了轉譯作用的效率以及準確性。在轉譯作用的起始階段中,許多信使RNA都使用一段夏因-達爾加諾序列使得核醣體得以辨識。夏因-達爾加諾序列通常坐落於起始密碼子 (AUG) 上游六個核苷酸的位置,並得以透過與16S 核醣體RNA上的夏因-達爾加諾序列互補序列互相鍵結進而穩定30S 核醣體次單元於信使RNA上。在30S次單元坐落於信使RNA後,便可於附近搜索適當的位置使得起始密碼子得以適當地位於核醣體P位點中。先前的研究已經了解轉譯起始因子(IF1, IF2 and IF3) 以及起始tRNA是如何參與在轉譯起始階段,然而信使RNA如何去徵調30S次單元以及30S次單元是如何於信使RNA上搜尋並到達夏因-達爾加諾序列的機制仍鮮為人知。在此,我們利用單分子螢光共振能量轉移技術進行研究,並展現了夏因-達爾加諾序列本身並不足以徵調核醣體。結合即時訊號的觀察,我們提出一個假說:30S次單元會先透過非專一性結合的方式坐落於轉譯起始位置兩側的序列上,進而移動至轉譯起始位置並完成轉譯起始階段。 | zh_TW |
dc.description.abstract | Translation initiation is a key step for regulating protein synthesis. As a rate limiting step, it controls not only translation efficiency but also fidelity. During translation initiation, many mRNAs employ a purine-rich Shine-Dalgarno (SD) sequence, which is usually located 6 nucleotides upstream of the start codon (AUG), to base pair with the anti-Shine-Dalgarno sequence (aSD) on 16S rRNA of the ribosomal 30S subunit. This SD-aSD interaction allows 30S subunits to bind to the mRNA stably and search locally for an appropriate start codon to locate in the P site of the 30S subunit. Many previous studies have shown how translation initiation factors (IF1, IF2 and IF3) and initiator tRNA participate in translation initiation. However, little is known about how the mRNA recruits 30S subunits and how 30S subunits move along the mRNA to the SD sequence and start site. Here, by using single molecule Förster Resonance Energy Transfer (smFRET), we demonstrated that the SD sequence alone was not sufficient to recruit ribosomes and either a long, single-stranded sequence or long, structured sequences could help recruit ribosomes. Combining real-time observations, we proposed that the 30S must first bind to flanking sequences nonspecifically and travel to the initiation start site to accomplish initiation. | en |
dc.description.provenance | Made available in DSpace on 2021-06-17T08:11:19Z (GMT). No. of bitstreams: 1 ntu-108-R06b43009-1.pdf: 4116942 bytes, checksum: a8db677068a6bf5a1ed2278f9cb368d2 (MD5) Previous issue date: 2019 | en |
dc.description.tableofcontents | CONTENTS
摘要………………………………………………………………………………………………………………………..ii Abstract ………………………………………………………………………………………………………………...…iii Figure list ………………………………………………………………………………………………………………..Viii CHAPTER 1 INTRODUCTION 1 1.1 TRANSLATION INITIATION 1 1.1.1 Shine-Dalgarno (SD) sequence 1 1.1.2 Start codon 2 1.1.3 Flanking sequences 2 1.1.4 Leaderless mRNA 3 1.2 MECHANISMS OF PROTEIN TRANSLOCATION ON NUCLEIC ACIDS 3 1.3 SINGLE-MOLECULE TECHNOLOGY 4 1.3.1 Definition 4 1.3.2 Single-molecule Förster Resonance Energy Transfer (FRET) 4 1.4 MOTIVE AND PURPOSE 5 CHAPTER 2 MATERIALS AND METHODS 6 2.1 MATERIALS 6 2.1.1 Buffers 6 2.1.2 Cell lines 7 2.1.3 Chemicals 7 2.1.4 Enzymes 9 2.1.5 Kits 10 2.1.6 Oligos 10 2.1.7 Constructs 12 2.2 METHODS 14 2.2.1 Polymerase Chain Reaction (PCR) 14 2.2.2 Plasmid Construction 14 2.2.3 Mutagenesis 16 2.2.4 Ribosome labeling 18 2.2.5 Slides and coverslip preparation for single-molecule experiments 19 2.2.6 Flow chamber assembling and sample preparation 20 CHAPTER 3 RESULTS 23 3.1 RIBOSOME RECRUITMENT 23 3.1.1 SD sequence is essential but not sufficient to recruit ribosomes 23 3.1.2 The length of the mRNA might play an important role in recruiting ribosomes. 23 3.1.3 Long structured upstream and downstream flanking sequences were able to recruit ribosomes. 24 3.2 RIBOSOME SEARCHING STRATEGY 25 3.2.1 Define FRET values for RBS binding and PIC conformation 25 3.2.2 Recruiting the 30S directly to the RBS seems unlikely. 26 3.2.3 The difference in initiation efficiency of GAA(52)-sRBS and GAA(2)-sRBS might correlate to the difference in binding rate. 26 3.2.4 The 0 FRET population in real-time observation represented a nonspecific binding state. 27 3.2.5 The nonspecifically binding 30S might travel to the ribosomal binding site through a sliding mechanism. 27 CHAPTER 4 DISCUSSION 29 4.1 RIBOSOME RECRUITMENT 29 4.1.1 Shine-Dalgarno sequence might be an essential element in translation initiation 29 4.1.2 The effect of the 30S platform on recruiting ribosomes 29 4.1.3 The effect of S1 on recruiting ribosomes 30 4.2 THE 30S DYNAMIC MOVEMENTS 31 4.2.1 The FRET distribution differed between Cy3-labeled 30S and Cy5-labeled 30S systems might be affected by protein induced fluorescence enhancement (PIFE). 31 4.2.2 Model 32 REFERENCES 33 APPENDIX 63 APPENDIX.A CALCULATION FOR BACKGROUND INDUCED FRET VALUE CHANGES 63 APPENDIX.B RECRUITING RIBOSOMES THROUGH RIBOSOMES IN PIC CONFORMATION 64 Figure content Fig. 1 Protein translocation mechanisms on nucleic acids. 38 Fig. 2 Total Internal Reflection Fluorescence 39 Fig. 3 Construction for pGAA(52) and pGAA(52)-sRBS. 40 Fig. 4 Construction for pGAA(6)-sRBS, pGAA(2)-sRBS and pS15hp1-sRBS. 41 Fig. 5 Construction for p1kb-GAA(6)-sRBS and pGAA(6)-sRBS-1kb... 42 Fig. 6 Flow chamber assembling. 43 Fig. 7 mRNA construct designs. 44 Fig. 8 Experimental designs. 45 Fig. 9 Initiation efficiency of different mRNA flanking sequences in 30S only condition. 46 Fig. 10 Conformational dynamics of the rpsO mRNA initiation start site. 47 Fig. 11 The FRET distribution of SP6-0 and GAA(52)-sRBS in 30S only and PIC conditions. 48 Fig. 12 The FRET distribution before and after wash in real-time observation. 50 Fig. 13 Real-time observation of the 30S binding to GAA(52)-sRBS. 51 Fig. 14 Real-time observation of the 30S binding to GAA(52). 52 Fig. 15 Real-time observation of the 30S binding to GAA(2)-sRBS. 53 Fig. 16 Background noise fluctuation calculation using Kinetic Analyzer. 54 Fig. 17 30S binding events determination using Kinetic Analyzer. 55 Fig. 18 The binding rate and dwell time distribution of different mRNA 56 Fig. 19 FRET distribution of different conditions in real-time experiment. 57 Fig. 20 Real-time traces in GAA(52)-sRBS showed interchanges between low FRET and high FRET. 58 Fig. 21 The hypothesis that the DNA-RNA hybrid structure might block the sliding of the 30S 59 Fig. 22 The FRET distribution of 30S only and PIC condition in Cy5 labeled 30S system 60 Fig. 23 Real-time traces of GAA(52)-sRBS in Cy5 labeled 30S system showed PIFE events. 61 Fig. 24 30S initiation model. 62 | |
dc.language.iso | en | |
dc.title | 以單分子螢光共振能量轉移技術研究核醣體在轉譯起始階段的搜尋機制 | zh_TW |
dc.title | The study of the 30S subunit searching mechanism in translation initiation by single-molecule FRET | en |
dc.type | Thesis | |
dc.date.schoolyear | 107-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 李弘文(Hung-Wen Li),李以仁(I-Ren Lee) | |
dc.subject.keyword | 轉譯作用起始,30S次單元,核醣體,轉譯起始位置,螢光共振能量轉移, | zh_TW |
dc.subject.keyword | Translation initiation,30S subunit,Ribosome,Flanking sequence,Initiation start site,Forster Resonance Energy Transfer, | en |
dc.relation.page | 64 | |
dc.identifier.doi | 10.6342/NTU201902817 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2019-08-16 | |
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 |
顯示於系所單位: | 分子與細胞生物學研究所 |
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