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研究生:許明倩
研究生(外文):Ming-Chien Hsu
論文名稱:藉由smFRET技術探討核醣體和mRNA在轉譯起始階段的動態結合反應
論文名稱(外文):Study of Dynamic Interaction between the Ribosome and mRNA at the Initiation Stage Using smFRET
指導教授:温進德
指導教授(外文):Jin-Der Wen
口試日期:2017-07-26
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:2017
畢業學年度:105
語文別:中文
論文頁數:74
中文關鍵詞:核醣體mRNA單分子螢光共振能量轉移SFP synthase酵素反應Shine-Dalgarno sequence
外文關鍵詞:ribosomemRNAsingle-molecule Förster resonance energy transferribosomal protein S5Shine-Dalgarno sequence
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在所有生物體內,核醣體為細胞內一重要胞器,從1953年結構陸續被解出,但是其轉譯作用機制仍須深入研究探討。在此我們的首要研究目的為觀察和mRNA之間的交互作用以及如何形成複合體。細菌的核醣體在進行轉譯時,小次單元30S會辨認mRNA上的核醣體結合位RBS (ribosome biding site) ,但是30S如何去辨認結合位的機制仍然不清楚,為了瞭解核醣體是直接辨認RBS,還是先和mRNA結合再滑行到RBS ? 所以我們利用螢光標記的方式,使用SFP synthase酵素系統的反應下在30S上標定螢光分子,然後藉由單分子螢光共振能量轉移技術 (smFRET) 偵測30S和mRNA之間的動態結合反應。
當原核生物在進行轉譯作用時,其mRNA上約六個鹼基的序列,同時也是核醣體結合位點,名稱為Shine-Dalgarno (SD) sequence。我們的研究目的想藉由smFRET觀察不同強度的SD sequence如何影響30S和mRNA之間的動態結合反應。從smFRET的分析可看到30S與不含SD、或極弱SD的mRNA只能短暫結合,進一步計算平均結合時間和機率,可以推測不同強度的SD sequence對30S結合mRNA之影響。另外也觀察30S如何辨認並且移動到RBS,我們發現當mRNA上有很強的RBS序列時,可以直接辨認並且穩定結合,但是分析smFRET的時間軌跡圖仍可發現有不同動態轉變的存在,顯示30S可能以滑動的方式在mRNA上尋找其結合位置。
Ribosomes are important machines for protein synthesis in all organisms. The structures of the ribosome have been revealed by TEM, X-ray and cryo-EM since its discovery in 1953. However, many aspects of the translation mechanism still remain unclear. Here we aim to investigate the interaction between mRNA and the ribosome during translation initiation by single-molecule techniques. For detection using single-molecule Förster resonance energy transfer (smFRET), we fluorescently label the small subunit 30S at the ribosomal protein S5 to probe the movement and dynamic interactions between the 30S and the mRNA. The binding of 30S and the mRNA is facilitated by the Shine-Dalgarno (SD) sequence which locates upstream of the start codon. We calculated the average binding times and the number of ribosome binding attempts for mRNA containing different strengths of SD sequences. It shows that the ribosome can only bind to the mRNA transiently if there are weak or no SD sequence. From among the various dynamics trajectories of FRET, we also found that the 30S may slide on the mRNA to reach the correct RBS. Further work needs to be dedicated toward this direction to uncover if the tRNA causes different dynamics while the ribosome slides on the mRNA.
審定書 i
致謝 ii
中文摘要 iii
ABSTRACT iv
目錄 v
圖目錄 viii
表目錄 x
縮寫檢索表 xi
第一章 導論 1
1.1 核醣體 1
1.1.1 基本介紹 1
1.1.2 轉譯作用 2
1.2 SFP synthase 4
1.3 Shine-Dalgarno sequence 4
1.4 單分子技術 5
1.4.1 基本介紹 5
1.4.2 螢光共振能量轉移 (Förster Resonance Energy Transfer) 6
1.5 研究動機和目的 6
第二章 材料與方法 8
2.1材料 8
2.1.1 勝任細胞品系 8
2.1.2 質體 8
2.1.3 試劑 9
2.1.4 藥品 9
2.1.5 酵素 11
2.1.6 引子序列 12
2.1.7 溶液 13
2.2方法 16
2.2.1 質體構築 16
2.2.2 大腸桿菌大量培養 17
2.2.3 核醣體純化 18
2.2.4 核醣體螢光標定及純化 20
2.2.5 聚合酶連鎖反應 (Polymerase Chain Reaction, PCR) 22
2.2.6 細胞外轉錄作用 (In vitro transcription) 23
2.2.7 DNA與RNA黏合反應 (Annealing reaction) 23
2.2.8 單分子螢光共振能量轉移實驗 (smFRET) 24
第三章 實驗結果 28
3.1 核醣體純化 28
3.2 核醣體螢光標定 28
3.3 質體構築之設計 30
3.4 DNA與RNA黏合反應 (Annealing reaction) 31
3.5 單分子螢光共振能量轉移實驗 (smFRET) 31
3.5.1 SD sequence的強弱和核醣體結合mRNA之關係 31
3.5.2 核醣體與mRNA之動態結合反應 33
第四章 討論 37
4.1 核醣體純化 37
4.2 核醣體螢光標定 37
4.3 SD sequence的強弱和核醣體結合mRNA之關係 38
4.3.1 時間軌跡圖觀測核醣體之動態 38
4.3.2 tRNA對於核醣體結合mRNA之影響 39
4.4 核醣體與mRNA之動態結合反應 39
4.5 總結 42
參考文獻 43

圖1、蔗糖濃度梯度製備 48
圖2、30S的核醣體蛋白質S5之示意圖 49
圖3、SFP synthase酵素系統反應 50
圖4、CoA-Cy5之HPLC純化 51
圖5、CoA-Cy3之HPLC純化 52
圖6、電泳凝膠遷移分析Cy5-30S和Cy3-30S 53
圖7、冷光素酶活性分析Cy5-30S 54
圖8、mGAA質體構築示意圖與序列 56
圖9、mGAA-sRBS質體構築示意圖與序列 58
圖10、DNA與RNA黏合反應設計 59
圖11、單分子共振能量轉移實驗材料與操作 60
圖12、電泳凝膠分析DNA與RNA黏合反應 61
圖13、核醣體結合mGAA之時間軌跡圖 62
圖14、Kinetic Analyzer軟體分析 63
圖15、核醣體結合mTTC2之時間軌跡圖 64
圖16、核醣體結合mGAA-sRBS之時間軌跡圖 65
圖17、mGAA-sRBS Pre-IC之時間軌跡圖 66
圖18、核醣體結合mHP1之時間軌跡圖 67
圖19、核醣體結合mHP1之時間軌跡圖 68
圖20、1 nM核醣體結合mGAA-sRBS之時間軌跡圖-無Salmon Sperm DNA 69
圖21、10 nM核醣體結合mGAA-sRBS之時間軌跡圖-無Salmon Sperm DNA 70
圖22、核醣體純化之OD值折線圖和RNA膠圖 71
圖23、核醣體純化步驟示意圖 72
圖24、結合時間分布圖 73
表1、核醣體結合mRNA的平均時間以及發生頻率 74
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