臺灣博碩士論文加值系統

在原核生物中，核醣體在轉譯起始的過程中必須先辨識mRNA 5’端未轉譯區(5’UTR)上的Shine-Dalgarno sequence (SD 序列)，進而使起始tRNA 配對到起始密碼子，轉譯方能開始。許多單股mRNA 在一般情況下會形成二級結構，在轉譯起始的過程中，這些二級結構必須被解開使密碼子能夠被呈現出來。大腸桿菌中rpsO 基因轉錄本5’UTR(rpsO 5’UTR)能夠藉由形成雙髮夾結構或偽結結構來調控自身序列的轉譯作用，但因為SD 序列被隱藏在雙髮夾結構中，所以核醣體只能結合在偽結結構開始轉譯作用。然而，在偽結結構只有SD 序列是暴露出來的，起始密碼子下游緊鄰另一個二級結構，所以核醣體必須解開下游的二級結構才能完成轉譯起始作用。

本篇研究經由單分子光鉗技術來探討當核醣體小次單元30S 及起始tRNA
存在時，對rpsO 5’UTR 的結構會有何影響。透過研究結果發現，和30S 反應過後的rpsO 5’UTR 和未加入30S 的組別，在光鉗實驗中看不出明顯差異。當同時有加入30S 及起始tRNA 的情況時，rpsO 5’UTR 下游的二級結構會被全部解開。刪減rpsO 5’UTR 兩個髮夾間的核苷酸數量，在光鉗實驗發現核醣體30S結合至RNA 的分子比例降低，在細胞體外轉譯實驗中得知刪減核苷酸數量的組別在轉譯效率上明顯下降。綜合以上結果，在轉譯起始階段30S 和起始tRNA 能夠解開rpsO 5’UTR 下游結構，且雙髮夾間的核苷酸扮演了30S 和起始tRNA 結合RNA 重要的角色。

In prokaryotes, ribosomes have to recognize the Shine-Dalgarno sequence (SDsequence) on mRNA 5’UTR, and then the initiator tRNA can pair to the start codon to initiate translation. Many mRNAs fold into secondary structures in normal situation.During the translation initiation process, these secondary structures have to be unfolded to revel the start codon. The rpsO gene transcript 5’ untranslated region (5’UTR) of Escherichia coli regulates its own translation through folding into a doublehairpin or a pseudoknot conformation. However, the SD sequence is concealed in the double-hairpin structure. Ribosomes can only bind to pseudoknot conformation to initiate translation. The SD sequence is fully exposed only on the pseudoknot. The start codon is adjacent to the downstream secondary structure, so the ribosome has to unwind the secondary structure to complete the initiation process.

In this research, we investigate the conformational change of the rpsO 5’ UTR in the presence of the ribosomal subunit 30S and initiator tRNA by using optical tweezers. Our results showed that there was no significant difference for the rpsO 5’UTR treated with or without 30S. In the presence of the 30S and initiator tRNA, rpsO 5’ UTR downstream secondary structure could be completely unfolded. The optical tweezers’ data showed that nucleotides deletion between the two hairpins decreased the 30S binding probability to RNA molecules, and also decreased the in vitro translation efficiency. These results demonstrate that the 30S and initiator tRNA can unfold the rpsO 5’ UTR downstream secondary structure, and the length of nucleotides between the two hairpins plays an important role in binding of 30S and tRNA to rpsO 5’ UTR.

口試委員會審定書 ii
致謝 iii
中文摘要 iv
ABSTRACT v
目錄 6
圖目錄 9
第一章 導論 11
1.1核醣體 11
1.1.1 功能 11
1.1.2 發現 11
1.1.3 組成 11
1.1.4 解旋能力 12
1.2轉譯 13
1.2.1 起始 13
1.2.2 延長 14
1.2.3 終止 14
1.3 rpsO基因 15
1.3.1 大腸桿菌(Escherichia coli)核醣體蛋白S15 15
1.3.2 核醣體蛋白S15的轉譯自體調節(Translational autoregulation) 15
1.4 單分子技術 (Single-Molecule Techniques) 16
1.4.1 介紹 16
1.4.2 光鉗 (Optical tweezers) 17
1.5 研究動機 17
第二章 材料與方法 18
2.1 材料 18
2.1.1 勝任細胞品系 18
2.1.2 質體 18
2.1.3 低聚物(Oligomers)及引子(Primers)設計 19
2.1.4 試劑組 21
2.1.5 藥品 22
2.1.6 酵素 23
2.1.7 儀器 24
2.1.8溶液 24
2.2 方法 27
2.2.1 載體構築 27
2.2.2 細胞外轉錄作用 ( in vitro transcription ) 29
2.2.3 聚合酶連鎖反應 (Polymerase Chain Reaction, PCR) 29
2.2.4 DNA handle與RNA之黏合反應 30
2.2.5 核醣體30S小次單元的純化 30
2.2.6 核醣體30S活性檢測 33
2.2.7 細胞外轉譯(in vitro Translation) 33
2.2.8 螢光素酶檢測(Luciferase assay) 33
第三章 結果 34
3.1 RNA樣本製備 34
3.2 核醣體小次單元30S對rpsO 5’UTR之影響 34
3.2.1 核醣體30S對野生型rpsO 5’ UTR之影響 35
3.2.1 核醣體30S對SD加強型rpsO 5’ UTR之影響 35
3.3 前轉譯起始複合體對rpsO 5’UTR之影響 36
3.4 SD序列和Stem 1之間的距離刪減對rpsO 5’UTR之影響 37
3.4.1 確認S15msSD-5nt之樣式 37
3.4.2 核醣體30S對S15msSD-5nt之影響 37
3.4.3 核醣體30S及起始tRNA對S15msSD-5nt之影響 38
3.4.4 雙髮夾結構間的核苷酸數量對轉譯起始的重要性 39
3.5 刪減rpsO 5’UTR雙髮夾間核苷酸數量對轉譯的影響 39
第四章 討論 40
4.1 rpsO 5’ UTR加入30S在光鉗實驗中觀察不出差異 40
4.2 S15msSD-5nt RNA的外力施予與否表現出的結構型態不同 40
4.3 未來實驗 41
參考文獻 42
圖1. pS15wt載體構築示意圖及部分序列示意圖 48
圖2. pS15wt-Rluc載體構築示意圖及部分序列示意圖 49
圖3. Insert序列更換限制酵素切位實驗設計示意圖 51
圖4. 光鉗實驗設置示意圖 52
圖5. 核醣體30S純化結果 53
圖6. 野生型rpsO 5'' UTR (S15wt)的結構、樣式比例及Force - extension分佈 55
圖7. 野生型rpsO 5'' UTR加入30S反應的樣式比例及Force - extension分佈 57
圖8. SD強化型rpsO 5'' UTR (S15msSD)結構示意圖 58
圖9. SD強化型rpsO 5'' UTR (S15msSD)的樣式比例及Force - extension分佈 59
圖10. SD強化型rpsO 5'' UTR 加入30S的樣式比例及Force - extension分佈 60
圖11. SD強化型rpsO 5'' UTR加入30S和起始tRNA的樣式、結構及Force - extension分佈 61
圖12. SD強化型rpsO 5'' UTR加入30S和起始tRNA後出現Stem 1開啟之力量範圍變大的分子 62
圖13. SD強化型rpsO 5 ''UTR 刪減5 nt (S15msSD-5nt)結構示意圖 64
圖14. SD強化型rpsO 5'' UTR 刪減5 nt的樣式及Force - extension分佈 65
圖15. SD強化型rpsO 5'' UTR 刪減5 nt加入30S的樣式及Force - extension分佈 66
圖16. 核醣體30S及起始tRNA充斥在S15msSD-5nt RNA會觀察到2T、1T及PK樣式交替出現 67
圖17. S15msSD-5nt摺疊的過程很可能是以雙髮夾結構出現 68
圖18. S15msSD、S15msSD-5nt細胞外轉譯效率圖 69

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