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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | ?進德(Jin-Der Wen) | |
dc.contributor.author | Yi-Lan Chen | en |
dc.contributor.author | 陳臆嵐 | zh_TW |
dc.date.accessioned | 2021-06-17T08:10:03Z | - |
dc.date.available | 2021-08-22 | |
dc.date.copyright | 2019-08-22 | |
dc.date.issued | 2019 | |
dc.date.submitted | 2019-08-16 | |
dc.identifier.citation | Aitken, C. E. and J. D. Puglisi (2010). 'Following the intersubunit conformation of the ribosome during translation in real time.' Nat Struct Mol Biol 17(7): 793-800.
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/73779 | - |
dc.description.abstract | 原核細胞中蛋白質的表現及恆定取決於轉譯起始的調控。轉譯起始時,核醣體會遇到擁有不同結構與序列的 mRNA。因此,了解這些不同的 mRNA 是如何與核醣體交互作用並正確地相嵌於核醣體上是一個極為重要的問題。在本研究中,透過利用單分子螢光共振能量轉移直接且即時的觀測核醣體及 mRNA 在轉譯起始時的交互作用。我們發現, mRNA 動態地來回纏繞核醣體 30S小次單元體。這個動態來回纏繞動作的頻率會因為遇到 mRNA的下游結構,像是雙股或髮夾結構,而增加。透過偵測下游結構被解開時所需要的力,我們發現,這樣的動態運動使得下游結構較不穩定。我們推測動態運動有助於核醣體有限地移動至正確的位置。透過較強的夏恩-達爾加諾序列形成的 mRNA 及核醣體交互作用可以幫助維持這樣的動態運動。
起始 tRNA的加入形成30S起始複合物 (30S IC),同時穩定 mRNA及幫助下游結構的解開至至多三個鹼基對。另外,無論起始tRNA存在與否,起始因子,尤其是 IF3 ,能幫助 mRNA 相嵌於核醣體上並抑制mRNA大幅度地運動。然而,IF3也有可能會降低 30S IC的穩定性。 在一些情況下,錯誤且穩定的 30S IC 會生成,尤其是下游出現雙股結構的時候。 這樣的雙股結構會發生在 sRNA 與 mRNA 結合時。 當這樣的情形發生時, IF3會透過移除起始轉移核醣核酸並分開mRNA及核醣體來拯救這個錯誤的複合物。在本研究中我們發現 mRNA 移動的彈性、起始密碼子及起始因子的選擇性及精確性,造就了轉譯初始發生的適切性。 | zh_TW |
dc.description.abstract | Regulation of translation initiation is important for protein synthesis and protein homeostasis in prokaryotic cells. During initiation, the ribosome encounters mRNAs with different sequences and structures. Therefore, understanding how different mRNAs correctly accommodate the ribosome and maintain the fidelity of translation at the beginning of initiation is essential. In this study, by directly observing the 30S ribosomal subunit and mRNA interaction by single-molecule FRET, we found the mRNA wraps the small subunit back and forth dynamically. This movement becomes more frequent if the downstream sequence forms structures, such as duplexes and hairpins. After measuring the unfolding force of downstream structures, we found the mRNA movement during initiation helps the ribosome destabilize downstream structures. We suggest that this dynamic movement helps the ribosome to search and find correct initiation site. Interaction between mRNA and the ribosome through strong Shine-Dalgarno sequence prevents the ribosome from dropping from the mRNA during the dynamic movement. Addition of initiator tRNA forms the 30S initiation complex (30S IC). Here, initiator tRNA suppresses the dynamic movement and opens downstream structures up to 3 bp. This step is a checkpoint for the correct initiation. Initiation factors, especially IF3, also stabilizes the mRNA before and after the initiator tRNA anchoring. However, IF3 also locally destabilize the 30S IC.
However, wrong stable 30S IC may form, especially when the downstream structure is a duplex structure. The duplex structures can be formed through small RNA (sRNA). In this case, IF3 can rescue the complex by removing the initiator tRNA and dissociating the mRNA-ribosome complex. Here, we reveal that the flexibility of mRNA movement, the selectivity of tRNA decoding, and the assurance of initiation factors help the ribosome maintain the robustness and fidelity of translation at the early initiation stage. | en |
dc.description.provenance | Made available in DSpace on 2021-06-17T08:10:03Z (GMT). No. of bitstreams: 1 ntu-108-D01b48004-1.pdf: 6679908 bytes, checksum: 8a3356bd0d8bd946fa26bb37da9e4cf5 (MD5) Previous issue date: 2019 | en |
dc.description.tableofcontents | 口試委員審定書 i
誌謝 ii 中文摘要 iii Abstract iv Contents vi Figure List xi Table List xiv Chapter 1. Introduction 1 1.1 Ribosome and translation in prokaryotes 1 1.2 Translation initiation process is heterogeneous 1 1.3 IF1 2 1.4 IF2 3 1.5 IF3 4 1.6 The 30S ribosomal subunit 5 1.7 The 50S ribosomal subunit 6 1.8 Initiator tRNA 7 1.9 mRNA 8 1.10 Leaderless mRNA 11 1.11 mRNA structures and translation initiation 12 1.12 Ribosome helicase activity 12 1.13 Single-molecule techniques 13 1.14 Single-molecule Förster Resonance Energy Transfer 13 1.15 Optical tweezers 14 1.16 Study of translation using single-molecule techniques 15 Chapter 2. Materials 17 2.1 Cell lines 17 2.2 Kits 17 2.2 Chemicals 18 2.3 Ladders 19 2.4 Enzymes 20 2.5 Primers 21 2.6 Primers for tweezers’ handles 22 2.7 Buffers 23 Chapter 3. Methods 25 3.1 Constructs for Tweezers’ experiments 25 3.2 Constructs for FRET experiments 25 3.3 Tweezers’ experiments-mRNA expression 26 3.4 Tweezers’ experiments-PCR 5’handle 26 3.5 Tweezers’ experiments-5’handle digoxigenin label 27 3.6 Tweezers’ experiments-3’handle biotin label 27 3.7 Tweezers’ experiments- Annealing of tethers 28 3.8 Tweezers’ experiments- Chamber preparation 28 3.9 Tweezers’ equipment setup 28 3.10 Calibration of miniTweezers 29 3.11 Tweezers’ experimental setup 30 3.12 Tweezers’ experiment setup for initiation complex 30 3.13 Data analysis for tweezers’ experiment 31 3.14 FRET experiments- Sample annealing 31 3.15 FRET experiments- Preparation of slides and coverslips 32 3.16 FRET equipment setup 33 3.17 Setup of FRET experiment 33 3.18 Data analysis for FRET experiments 34 3.19 Purification of Initiation Factors 35 3.20 Purification of 70S ribosomes 36 3.21 Purification of 30S and 50S ribosomal subunits 37 3.22 Testing ribosome activity 37 3.23 Purification of S100DEAE 38 3.24 Charging of tRNAfMet 38 Chapter 4. Results 40 4.1 Aims 40 4.2 Design of the single-molecule FRET experiment 40 4.3 Unstructured mRNA is compacted in Mg2+ buffer 41 4.4 The unstructured mRNA accommodated on the 30S PIC, 30S IC and 70S IC has the same EFRET value 42 4.5 Small dynamic movement has been found in 30S PIC state 42 4.6 Shine-Dalgarno sequence is important for the 30S subunit binding 43 4.7 Hairpin structured mRNA in each state 44 4.8 Hairpin structured mRNA shows different dynamic patterns after the 30S subunit binding 44 4.9 Dynamic movement is not a drop off and rebind event 45 4.10 mRNA with downstream duplex structure has the same dynamic movement as the mRNA with downstream hairpin structure 46 4.11 More single-stranded region than the ribosome footprint is required for better translation initiation 47 4.12 Stable 30S IC formed with downstream duplex structure 48 4.13 Moving the hairpin near to the AUG start codon shows less 30S IC formation 49 4.14 The protein induced fluorescence enhancement is observed when measuring the construct with closer hairpin structure 50 4.15 Decoding is important for stabilizing the mRNA accommodation 51 4.16 Initiation factors stabilize the 30S PIC with F+18 51 4.17 Initiation factors, especially the IF3, stabilize the 30S PIC with F+11hp 52 4.18 The initiation factors stabilize the 30S IC complex with F+11hp 53 4.19 The initiation factors also stabilize the 30S PIC with F+8 54 4.20 F+8 30S IC could be rescued by initiation factors 55 4.21 Initiation factor F+8hp:H20 56 4.23 The force and size changes of T+11hp 59 4.24 Hairpins with higher stability are used for testing the influence of the downstream structure to the ribosome 60 4.25 Testing if the bulge position influences the stability of downstream structure 61 4.26 Designing a construct which can measure binding signal of the 30S subunit 63 4.27 Testing how many base pairs could be opened by the 30S IC 64 4.28 Move the SD sequence near to the AUG start codon to see how the ribosome binding site influences the downstream structure 65 Chapter 5. Discussion 67 5.1 Models 67 5.2 Shine-Dalgarno sequence is a prerequisite for mRNA accommodation 67 5.3 Use dnaX hairpin as the model hairpin 68 5.4 Prevalence of the mRNA structure in E. coli 68 5.5 Helicase activity passive or active 69 5.6 Decoding of the initiator tRNA might close the mRNA entry site 70 5.7 Dynamic movement 70 5.8 PIFE phenomenon near the surface of the narrow mRNA channel 71 5.9 Initiation factor effect 72 5.10 Wrong complexes we detected can be a case of siRNA, miRNA or sRNA in natural 72 5.11 Other rescue systems 73 5.12 Dynamic of hairpins or structures 73 5.13 Translation initiation in Eukaryotes 73 5.14 Conclusions 74 References 75 Appendix I. Fluctuated Intensity after 70S IC Formation 156 AI.1 Specific Aims 156 AI.2 Experimental setup 156 AI.3 The EFRET versus time traces show large fluctuations 157 AI.4 Cy3 dye at different positions have various fluctuation patterns 158 AI.5 The fluctuation is not related to the ratchet movement 159 AI.6 Discussion 161 Appendix II. Computational Modeling of polysome effect on dnaX -1 programmed ribosomal frameshifting 162 AII.1 Abstract 162 AII.2 Introduction 162 Quantitative biology 162 Recoding 163 dnaX -1 programmed ribosomal frameshifting 164 Preliminary data and Polysome effect 164 Specific Aims 165 AII.3 Results 166 Rationale 166 Regular modeling 167 Random modeling 170 AII.4 Discussion 172 Meanings of regular and random modeling results 172 R0 and translation initiation strength 173 Figure List Figure 3.1 Plasmid map for pT7SP6 90 Figure 3.2 Plasmid map for pS15WT 91 Figure 3.3 Plasmid map for pSP6 92 Figure 4.1 Experimental design for smFRET experiments 93 Figure 4.2 Materials needed for each initiation state 94 Figure 4.3 Sequences of the constructs we used for smFRET experiment 95 Figure 4.4 Construct lineage for smFRET experiment 96 Figure 4.5 Result of the unstructured mRNA construct, F+18 98 Figure 4.6 The high EFRET measured is because of the Mg2+ effect 99 Figure 4.7 vbFRET analysis of the unstructured mRNA, F+18 100 Figure 4.8 Results of the unstructured mRNA with weak SD sequence, FwSD+18 101 Figure 4.9 Results of the unstructured mRNA with SD-less sequence, FaSD+18 102 Figure 4. 10 Results of structured mRNA construct, F+11hp 103 Figure 4.11 Increase the temperature helps the 30S IC formation with F+11hp 104 Figure 4.12 vbFRET analysis of the structured mRNA, F+11hp 105 Figure 4.13 Testing if the FRET spikes are drop off and rebind events 106 Figure 4.14 Experiment design with the biotin-30S 107 Figure 4.15 Results of the biotin-30S experiment with F+11hp 108 Figure 4.16 Construct design for experiments with different duplex positions 109 Figure 4.17 Results for testing duplex structure at different positions 111 Figure 4.18 Same results found when using RNA-DNA and RNA-RNA duplexes 112 Figure 4.19 Annealing test for F+8:H12 construct 113 Figure 4.20 Result for F+8:H12 114 Figure 4.21 Experimental design for testing the influence of hairpin structures at different position 115 Figure 4.22 Results of F+8hp and F+3hp 116 Figure 4.23 Protein induced fluorescence enhancement found in F+3hp and F+8hp 117 Figure 4.24 Testing the decoding efficiency using Fuuu+11hp 118 Figure 4.25 The initiation factors, both IF1 and IF3 stabilize the dynamic movement in 30S PIC state 119 Figure 4.26 The influence of translation initiation factors to structure mRNA, F+11hp 121 Figure 4.27 The influence of initiation factors to the duplex construct, F+8 122 Figure 4.28 Testing the dropping rate of F+8 30SPIC with IF3 after buffer washing 124 Figure 4.29 Time course EFRET histogram of the F+8 30S IC after buffer washing with or without the IF3 125 Figure 4.30 Experimental design for testing the structure in the middle of the ribosome binding site 126 Figure 4.31 The influence of translation initiation factors to structure mRNA, F+8hp:H20 128 Figure 4.32 Experimental setup for studies using optical tweezers 129 Figure 4.33 The sequences of constructs used for optical tweezers’ experiment 130 Figure 4.34 Predicted structures the hairpins used in optical tweezers’ experiment 131 Figure 4.35 Construct lineage for optical tweezer’s experiments 132 Figure 4.36 F-X distribution plot for T+11hp 134 Figure 4.37 Histogram of unfolding force and hairpin size in each state with T+11hp 135 Figure 4.38 F-X distribution plot for T+11hpM 136 Figure 4.39 Histograms of unfolding force and hairpin size in each state with F+11hpM 137 Figure 4.40 F-X distribution plot for T+11hpY 138 Figure 4.41 Histogram of unfolding force and hairpin size in each state with T+11hpY 139 Figure 4.42 F-X distribution plot for T+11hpE 140 Figure 4.43 Histograms of unfolding force and hairpin size in each state with T+11hpE 141 Figure 4.44 F-X traces before and after the 30S subunit binding 142 Figure 4.45 F-X distribution plot for T+11hpE2 143 Figure 4.46 Histograms of unfolding force and hairpin size in each state with T+11hpE2 144 Figure 4.47 Differences of the unfolding force and hairpin size of each construct in each state 145 Figure 4.48 Comparing the force distributions of 30S PIC and mRNA states of each construct 146 Figure 4.49 Testing how many base pairs could be opened after the 30S IC formation 147 Figure 5.1 Models of the formation of 30S IC with various mRNA conformations 149 Figure 5.2 Conclusions 150 Figure AI.1 Experimental design for measuring the influence of 70S IC to downstream structure 157 Figure AI.2 Cy3 intensity shows large fluctuation after the 70S IC formed 158 Figure AI. 3 Cy3 dye that near the initiation site interact with the ribosome 159 Figure AI. 4 The Fluctuation is not related to the ratchet movement 160 Figure AII.1 Preliminary data 165 Figure AII.2 Regular modeling 168 Figure AII.3 Random modeling 172 Table List Table 1. Percentages and major EFRET peaks of mRNA in various ribosome-bound states 151 Table 2. Unfolding forces and sizes of the mRNA hairpins in various ribosome-bound state 154 | |
dc.language.iso | en | |
dc.title | 核醣體與訊息核醣核酸動態相嵌可促進轉譯起始過程 | zh_TW |
dc.title | Dynamic Accommodation of mRNA onto Ribosomes Facilitates Translation Initiation | en |
dc.type | Thesis | |
dc.date.schoolyear | 107-2 | |
dc.description.degree | 博士 | |
dc.contributor.oralexamcommittee | 張功耀(Kung-Yao Chang),李弘文(Hung-Wen Li),李以仁(I-Ren Lee),周信宏(Hsin-Hung David Chou) | |
dc.subject.keyword | 轉譯起始,mRNA相嵌,轉譯起始因子,核醣體,單分子,光鉗,單分子螢光共振能量轉移, | zh_TW |
dc.subject.keyword | translation initiation,mRNA accommodation,initiation factor 3,ribosome,single-molecule,smFRET,optical tweezers, | en |
dc.relation.page | 174 | |
dc.identifier.doi | 10.6342/NTU201902818 | |
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 | 基因體與系統生物學學位學程 | zh_TW |
顯示於系所單位: | 基因體與系統生物學學位學程 |
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