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標題: | 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 Study of Dynamic Interaction between the Ribosome and mRNA at the Initiation Stage Using smFRET |
作者: | Ming-Chien Hsu 許明倩 |
指導教授: | ?進德(Jin-Der Wen) |
關鍵字: | 核醣體,mRNA單分子螢光共振能量轉移,SFP synthase酵素反應,Shine-Dalgarno sequence, ribosome,mRNA,single-molecule Forster resonance energy transfer,ribosomal protein S5,Shine-Dalgarno sequence, |
出版年 : | 2017 |
學位: | 碩士 |
摘要: | 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. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/67071 |
DOI: | 10.6342/NTU201702917 |
全文授權: | 有償授權 |
顯示於系所單位: | 分子與細胞生物學研究所 |
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