臺灣博碩士論文加值系統

核醣體是生物製造蛋白質的工廠。一條mRNA上通常有多個核醣體同時進行轉譯，稱作聚核醣體。核醣體在轉譯過程中遇到特殊的mRNA序列時會發生框架位移，從一段mRNA序列轉譯出不同的蛋白質產物。大腸桿菌的dnaX基因框架位移位點由滑動序列、上游的內部SD序列和下游的髮夾結構三個部分組成。當核醣體轉譯到滑動序列時，受到下游髮夾結構的阻礙和上游SD序列的拉扯，有機會往上游方向滑動，從0轉譯框架進入-1轉譯框架。兩個轉譯框架分別做出DNA聚合酶III的γ和τ次單元。
本文在大腸桿菌內研究聚核醣體現象對轉譯框架位移的影響。我們用dnaX轉譯框架位移位點作為材料，通過西方墨點法定量兩個轉譯框架的蛋白質，進而計算出框架位移比例。我們發現核醣體在遇到SD序列和二級結構時轉譯速度減慢，可能會被上游的核醣體追上。此時，下游核醣體代替原本框架位移位點的髮夾結構，促使上游核醣體發生-1框架位移。如果去掉下游的SD序列或二級結構，使核醣體不再聚集，框架位移比例都會下降。另外，內部SD序列還可以抓住游離的30S次單元，同樣能夠阻礙上游核醣體引起轉移框架位移。通過這些研究，我們成功觀測到聚核醣體中相鄰核醣體會互相影響，並且對轉譯結果產生顯著的影響。

The ribosome is a ubiquitous protein manufactory for all living creatures. There are usually copious ribosomes, also called polyribosome, co-translating on one mRNA. The ribosome has a chance to shift its reading frame while encountering a special mRNA sequence, like the frameshifting site of dnaX gene in Escherichia coli, and thus translates the downstream mRNA sequence to another different protein product. The dnaX frameshifting site consists of a slippery sequence, an upstream internal Shine-Dalgarno sequence and a downstream hairpin. Because of being obstructed by hairpin and drawn by the SD sequence, the ribosome may shift one base towards upstream on the slippery sequence from the 0 frame to -1 frame. As a consequence, different codons on two frames are respectively translated into γ and τ subunits of DNA polymerase III.
In this thesis, we investigate how polyribosome affects translation frameshifting in E. coli in vivo. We use the dnaX frameshifting site as a model, quantify protein products from two frames by Western blot, and calculate frameshifting efficiency. We find that a ribosome can be temporarily stalled by a strong SD sequence or secondary structure and then caught up by following ribosomes. At this time, the stalled ribosome may mimic the role of the hairpin and promote the following ribosome to shift to the -1 frame on the slippery sequence. When we mutate the downstream SD sequence or secondary structure, ribosomes are unable to accumulate, resulting in a reduction of frameshifting efficiency. Moreover, not only an elongating ribosome but also a free 30S ribosomal subunit can trigger upstream ribosomes frameshifting by binding to the internal SD sequence. Through our research, we have successfully observed the interaction between ribosomes, which may remarkably affect translation outcome.

口試委員會審定書 i
致謝 ii
摘要 iii
ABSTRACT iv
目錄 v
圖目錄 viii
表目錄 ix
英文縮寫檢索表 x
第一章 前言 1
1.1 蛋白質 1
1.2 核醣體與轉譯 1
1.3 轉運RNA 2
1.4 原核生物轉譯過程 2
1.5 聚核醣體 4
1.6 擺動配對 4
1.7 框架位移 5
1.8 聚核醣體抑制mRNA二級結構的形成 5
1.9 不同框架終止密碼子引起不同框架位移比例 6
1.10 研究目的和動機 7
第二章 材料和方法 9
2.1 載體 9
2.2 菌株 9
2.3 酵素 9
2.4 抗體 9
2.5 試劑 9
2.6 藥品 10
2.7 主要耗材 10
2.8 主要儀器 11
2.9 聚丙烯醯胺凝膠 11
2.10 緩衝液 11
2.11 勝任細胞製備 12
2.12 質體構建 12
2.13 蛋白質表達 12
2.14 細胞外實驗 13
2.15 西方墨點法 13
2.16 蛋白質定量和分析 14
第三章 結果 15
3.1 模仿p1FS和p1FI的距離設計實驗 (pFsΔhp-47/48/50-St) 15
3.2 滑動序列到終止密碼子的距離減半 (pFsΔhp-17/20/23-St) 16
3.3 微調滑動序列到-1終止密碼子的距離 (pmonoFSI, pmonoFSI-9) 17
3.4 改用0框架終止密碼子暫停核醣體 (pFsΔhp-18-St) 18
3.5 減少mRNA的數量以增加核醣體的密度 (pFsΔhp-47/48/23-St) 19
3.6 減少RF1的濃度來促進核醣體聚集 (pFsΔhp-48-St) 20
3.7 終止密碼子下游再加入髮夾結構 (pFsΔhp-48-St-Hp/eHp) 20
3.8 加入髮夾結構並且讓距離減半 (pFsΔhp-18/21/24-St-Hp) 21
3.9 用SD序列和髮夾結構促進核醣體相碰 (pFsΔhp-4/5/6/7-Sd-Hp) 22
3.10 驗證框架位移和下游SD序列的關聯性 (pFsΔhp-5-msd-Hp) 23
3.11 驗證框架位移和下游髮夾結構的關聯性 (pFsΔhp-47-St) 23
3.12 驗證框架位移和聚核醣體的關聯性 (pRt-FsΔhp-5-Sd/msd-Hp) 23
第四章 討論 26
4.1 終止密碼子對轉譯的影響 26
4.2 髮夾結構對轉譯和框架位移的影響 26
4.3 內部SD序列對轉譯和框架位移的影響 28
4.4 影響核醣體聚集的其他因素 28
4.5 聚核醣體現象對轉譯起始的影響 29
4.6 核醣體聚集導致的不均勻分佈以及產生的影響 30
4.7 聚核醣體情況下每個核醣體覆蓋的mRNA長度 31
4.8 實驗方法改進 32
4.9 後續研究 32
參考文獻 36
圖1. 核醣體 40
圖2. 轉譯過程(Moore 2012) 41
圖3. 聚核醣體(Slayter, Warner et al. 1963) 42
圖4. 鹼基配對 43
圖5. 框架位移 44
圖6. 原始載體pGS-21a 45
圖7. 質體ptriFS 46
圖8. 質體p1FS與p1FI 47
圖9. 模仿p1FS與p1FI並去掉髮夾結構(pFsΔhp-47/48/50-St) 48
圖10. 滑動序列與終止密碼子之間的距離減半(pFsΔhp-17/20/23-St) 49
圖11. 微調滑動序列到終止密碼子的距離(pmonoFSI, pmonoFSI-9) 50
圖12. 改用0框架終止密碼子暫停核醣體(pFsΔhp-18-St) 51
圖13. 減少mRNA數量進行in vitro實驗(pFsΔhp-47/48/23-St) 52
圖14. 減少RF1的濃度進行in vitro實驗(pFsΔhp-48-St) 53
圖15. 加入髮夾結構幫助終止密碼子暫停核醣體(pFsΔhp-48-St-Hp/eHp) 54
圖16. 加入髮夾結構並且連接片段距離減半(pFsΔhp-18/21/24-St-Hp) 55
圖17. 用下游SD序列和髮夾結構聚集核醣體(pFsΔhp-4/5/6/7-Sd-Hp) 56
圖18. 改變下游SD序列(pFsΔhp-5-msd-Hp) 57
圖19. 同時去掉下游SD序列與髮夾結構(pFsΔhp-47-St) 58
圖20. 加入聯讀序列(pRt-FsΔhp-5-Sd-Hp) 59
圖21. 下游SD序列抓住游離30S次單元示意圖 60
圖22. 改變下游SD序列同時加入聯讀序列(pRt-FsΔhp-5-msd-Hp) 61
圖23. 主要實驗結果數據對比 62
表1. 各質體插入片段DNA序列 63
表2. 各質體蛋白質產物大小計算值 67
表3. 滑動序列下游不同距離加入SD序列的實驗數據分析 68
表4. 衍伸自pFsΔhp-5-Sd-Hp的相關實驗數據分析 69

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