跳到主要內容

臺灣博碩士論文加值系統

(3.235.56.11) 您好!臺灣時間:2021/07/29 09:50
字體大小: 字級放大   字級縮小   預設字形  
回查詢結果 :::

詳目顯示

我願授權國圖
: 
twitterline
研究生:張舒雅
研究生(外文):Shu-Ya Chang
論文名稱:利用單分子螢光共振能量轉移技術探討核醣體在rpsO基因轉錄本上對轉譯起始的影響
論文名稱(外文):Observing Translation Initiation of the Ribosome on rpsO Transcript by smFRET
指導教授:温進德
指導教授(外文):Jin-Der Wen
口試委員:張功耀黃筱鈞
口試委員(外文):Kung-Yao ChangHsiao-Chun Huang
口試日期:2015-07-15
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:2015
畢業學年度:103
語文別:中文
論文頁數:75
中文關鍵詞:核醣體轉譯起始作用單分子螢光共振能量轉移技術rpsO基因
外文關鍵詞:ribosometranslation initiationsingle-moleculeFRETrpsO gene
相關次數:
  • 被引用被引用:0
  • 點閱點閱:41
  • 評分評分:
  • 下載下載:0
  • 收藏至我的研究室書目清單書目收藏:0
許多mRNA在一般情況下會形成二級結構,然而,當轉譯作用進行時,其二級結構必須被解開使得密碼子能夠被呈現出來。先前研究指出核醣體自己本身在轉譯作用進行時即具有解旋酶(helicase)的活性,可以解開mRNA所形成的二級結構。大腸桿菌中rpsO基因轉錄本的5’未轉譯區(5’UTR)能夠藉由形成假結或雙髮夾結構來調控其本身的轉譯作用,但因為雙髮夾結構的SD(Shine-Dalgarno)序列無法被呈現,所以核醣體只能與假結結構結合並開始轉譯作用的進行。除此之外,在假結結構中只有SD序列是暴露出來的,而起始密碼子下游卻緊鄰二級結構,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。然而,目前對於假結結構在起始作用中哪個階段被核醣體解開的仍然不清楚。
本篇研究透過單分子螢光共振能量轉移技術來探討當核醣體小次單元及起始tRNA存在時,rpsO基因轉錄本之5’未轉錄區的結構會有何變化。研究結果發現,當核醣體小次單元存在並與mRNA結合時,mRNA形成假結結構的比例會增加,與先前研究核醣體小次單元只能和假結結構結合的結果相符。不只如此,當30S結合到假結結構的SD序列上時,核醣體小次單元能夠藉由解開部分mRNA的二級結構來找到起始密碼子AUG。而當同時有30S及起始tRNA出現時,起始tRNA能夠幫助核醣體小次單元將下游的二級結構全部解開,使得前轉譯起始複合體可以完成。以上研究結果顯示,在轉譯起始階段,核醣體小次單元自己本身即可表現解旋酶活性,將具有二級結構的mRNA解開,讓轉譯作用可以順利進行。


Many mRNAs fold into secondary structures; however, their codons must be in single-stranded form to be translated. Previous research has revealed that the ribosome itself has helicase activity during the translation process. The rpsO gene transcript of Escherichia coli regulates its own translation through the 5’ untranslated region (5’ UTR), which can fold into a pseudoknot or a double-hairpin conformation. The two structures can be interchanged spontaneously, but the ribosome can only bind to the pseudoknot to initiate translation. On the pseudoknot, only the Shine-Dalgarno (SD) sequence but not the AUG start codon is fully exposed, so the ribosome has to unwind part of the secondary structure to complete the initiation. However, it remains unclear in which
stage the conformation is opened by the ribosome.
In this study, we characterize the conformational change of the rpsO 5’ UTR in the presence of the 30S ribosomal subunit and initiator tRNA (charged formyl-methionyl tRNA) by using single-molecule fluorescence resonance energy transfer (smFRET). Our results show that the population of the pseudoknot form is increased when 30S binds to the RNA. 30S would begin to search for the AUG start codon after binding to the SD sequence of the pseudoknot by partially unwinding the local structures. In the presence of the initiator tRNA, 30S may completely unwind a stem of the pseudoknot and form the pre-initiation complex. These results demonstrate that the 30S ribosomal subunit alone can perform its helicase activity during the initiation stage.


目錄
口試委員審定書 ii
致謝 iii
中文摘要 iv
ABSTRACT v
目錄 vi
圖目錄 ix
表目錄 x
第一章 導論 1
1.1 核醣體 1
1.1.1 功能 1
1.1.2 結構 1
1.1.3 解旋作用 2
1.2 轉譯作用 3
1.2.1 起始階段 3
1.2.2 延長階段 4
1.2.3 終止階段 4
1.3 rpsO基因 5
1.4 單分子技術 6
1.4.1 簡介 6
1.4.2 螢光共振能量轉移 (fluorescence resonance energy transfer ) 6
1.5 研究動機 7
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 8
2.1.4 藥品 9
2.1.5 酵素 10
2.1.6 載體構築序列及引子設計 10
2.1.7 溶液 12
2.2 方法 14
2.2.1 載體構築及純化 14
2.2.2 聚合酶連鎖反應 (Polymerase Chain Reaction, PCR) 14
2.2.3 細胞外轉錄作用 ( in vitro transcription ) 15
2.2.4 細胞外轉譯作用 ( in vitro translation ) 15
2.2.5 DNA引子及RNA黏合反應 16
2.2.6 核醣體30S小次單元的純化 16
2.2.7 單分子螢光共振能量轉移實驗 18
第三章 結果 21
3.1 RNA樣本的製備 21
3.2 確認rpsO基因結構的FRET效率 21
3.2.1 雙髮夾結構的FRET效率 21
3.2.2 假結結構的FRET效率 22
3.2.3 rpsO基因轉錄本5’UTR的FRET效率 22
3.3 核醣體小次單元30S對rpsO基因轉錄本的影響 23
3.4 核醣體30S小次單元解開假結結構時的FRET效率 24
3.5 起始tRNA能協助30S小次單元完整解開二級解構 25
3.6 進一步確認30S和起始tRNA與rpsO基因轉錄本結構變化的關係 26
3.6.1 加強SD序列可穩定30S與RNA的結合 26
3.6.2 去除起始密碼子AUG對轉譯起始作用的影響 27
第四章 討論 29
4.1 解開二級結構的能量來源 29
4.2 核醣體30S小次單元具有解旋能力的部位 29
4.3 核醣體蛋白S15對rpsO基因轉錄本在轉譯起始作用的影響 30
4.4 rpsO基因轉錄本的5’ UTR形成雙髮夾結構的功能 31
4.5 起始tRNA協助30S解開二級結構的作用機制 32
4.6 核醣體30S小次單元的解旋能力與解旋酶的異同 33
4.7 未來展望 34
參考文獻 35

圖目錄
圖1. pS15WT-s載體構築示意圖及部分序列示意圖 41
圖2. 核醣體純化之結果 42
圖3. 單分子螢光共振能量轉移實驗使用之RNA轉錄本 43
圖4. 單分子螢光共振能量轉移實驗設置 44
圖5. rpsO基因轉錄本5’UTR結構與DNA探針之相對位置示意圖 45
圖6. 雙髮夾結構(mHPGC)序列及FRET與分子螢光強度關係圖 47
圖7. 雙髮夾結構(mHPGC)的FRET效率及時間軌跡圖 49
圖8. 假結結構(dAC)序列及FRET與分子螢光強度關係圖 51
圖9. 假結結構(dAC)的FRET效率及時間軌跡圖 53
圖10. rpsO 5’UTR的FRET與分子螢光總強度及FRET效率示意圖 55
圖11. rpsO基因轉錄本5’UTR的時間軌跡圖 57
圖12. 30S存在時5’UTR的FRET與分子螢光總強度及轉換效率示意圖 58
圖13. 核醣體30S存在時rpsO 5’UTR的時間軌跡圖 60
圖14. mHP1序列及FRET與分子螢光強度關係圖 61
圖15. mHP1的FRET效率及時間軌跡圖 62
圖16. 起始tRNA與30S同時存在時5’UTR的FRET與分子螢光總強度及轉換效率示意圖 64
圖17. 起始tRNA與30S同時存在時rpsO 5’UTR的時間軌跡圖 66
圖18. 強SD結構(mPKsSD)序列及FRET與分子螢光強度關係圖 67
圖19. 強SD結構(mPKsSD)的FRET效率及時間軌跡圖 68
圖20. 去除AUG結構(mdAUG)序列及FRET與分子螢光強度關係圖 69
圖21. 去除AUG結構(mdAUG)的FRET效率及時間軌跡圖 70
圖22. 細胞外轉譯作用實驗設計及結果 71
圖23. 核醣體蛋白S15與rpsO 5’UTR結合位置示意圖 72
圖24. 核醣體蛋白S15存在時rpsO 5’UTR的時間軌跡及FRET效率圖 73

表目錄
表1. 實驗中使用的所有RNA結構之序列 74
表2. 每個結構在各實驗條件下的FRET效率 75



Agalarov, S. C., G. Sridhar Prasad, P. M. Funke, C. D. Stout and J. R. Williamson (2000). "Structure of the S15,S6,S18-rRNA complex: assembly of the 30S ribosome central domain." Science 288(5463): 107-113.
Aitken, C. E., R. A. Marshall and J. D. Puglisi (2008). "An oxygen scavenging system for improvement of dye stability in single-molecule fluorescence experiments." Biophys J 94(5): 1826-1835.
Ban, N., P. Nissen, J. Hansen, P. B. Moore and T. A. Steitz (2000). "The complete atomic structure of the large ribosomal subunit at 2.4 A resolution." Science 289(5481): 905-920.
Berk, V., W. Zhang, R. D. Pai and J. H. Cate (2006). "Structural basis for mRNA and tRNA positioning on the ribosome." Proc Natl Acad Sci U S A 103(43): 15830-15834.
Blattner, F. R., G. Plunkett, 3rd, C. A. Bloch, N. T. Perna, V. Burland, M. Riley, J. Collado-Vides, J. D. Glasner, C. K. Rode, G. F. Mayhew, J. Gregor, N. W. Davis, H. A. Kirkpatrick, M. A. Goeden, D. J. Rose, B. Mau and Y. Shao (1997). "The complete genome sequence of Escherichia coli K-12." Science 277(5331): 1453-1462.
Carter, A. P., W. M. Clemons, D. E. Brodersen, R. J. Morgan-Warren, B. T. Wimberly and V. Ramakrishnan (2000). "Functional insights from the structure of the 30S ribosomal subunit and its interactions with antibiotics." Nature 407(6802): 340-348.
Carter, A. P., W. M. Clemons, Jr., D. E. Brodersen, R. J. Morgan-Warren, T. Hartsch, B. T. Wimberly and V. Ramakrishnan (2001). "Crystal structure of an initiation factor bound to the 30S ribosomal subunit." Science 291(5503): 498-501.
Dahlquist, K. D. and J. D. Puglisi (2000). "Interaction of translation initiation factor IF1 with the E. coli ribosomal A site." J Mol Biol 299(1): 1-15.
Forster, T. (1948). "Zwischenmolekulare Energiewanderung und Fluoreszenz." Annalen der Physik 437: 55-75.
Frank, J., J. Zhu, P. Penczek, Y. Li, S. Srivastava, A. Verschoor, M. Radermacher, R. Grassucci, R. K. Lata and R. K. Agrawal (1995). "A model of protein synthesis based on cryo-electron microscopy of the E. coli ribosome." Nature 376(6539): 441-444.
Gualerzi, C., G. Risuleo and C. L. Pon (1977). "Initial rate kinetic analysis of the mechanism of initiation complex formation and the role of initiation factor IF-3." Biochemistry 16(8): 1684-1689.
Gualerzi, C. O. and C. L. Pon (1990). "Initiation of mRNA translation in prokaryotes." Biochemistry 29(25): 5881-5889.
Ha, T. (2001). "Single-molecule fluorescence resonance energy transfer." Methods 25(1): 78-86.
Kapanidis, A. N. and T. Strick (2009). "Biology, one molecule at a time." Trends Biochem Sci 34(5): 234-243.
Keller, E. B., P. C. Zamecnik and R. B. Loftfield (1954). "The role of microsomes in the incorporation of amino acids into proteins." J Histochem Cytochem 2(5): 378-386.
Kennell, D. and H. Riezman (1977). "Transcription and translation initiation frequencies of the Escherichia coli lac operon." J Mol Biol 114(1): 1-21.
Kisselev, L. L. and R. H. Buckingham (2000). "Translational termination comes of age." Trends Biochem Sci 25(11): 561-566.
La Teana, A., C. O. Gualerzi and R. Brimacombe (1995). "From stand-by to decoding site. Adjustment of the mRNA on the 30S ribosomal subunit under the influence of the initiation factors." RNA 1(8): 772-782.
Lake, J. A. (1976). "Ribosome structure determined by electron microscopy of Escherichia coli small subunits, large subunits and monomeric ribosomes." J Mol Biol 105(1): 131-139.
Laursen, B. S., H. P. Sorensen, K. K. Mortensen and H. U. Sperling-Petersen (2005). "Initiation of protein synthesis in bacteria." Microbiol Mol Biol Rev 69(1): 101-123.
Liljas, A. and S. al-Karadaghi (1997). "Structural aspects of protein synthesis." Nat Struct Biol 4(10): 767-771.
Lingelbach, K. and B. Dobberstein (1988). "An extended RNA/RNA duplex structure within the coding region of mRNA does not block translational elongation." Nucleic Acids Res 16(8): 3405-3414.
Marzi, S., A. G. Myasnikov, A. Serganov, C. Ehresmann, P. Romby, M. Yusupov and B. P. Klaholz (2007). "Structured mRNAs regulate translation initiation by binding to the platform of the ribosome." Cell 130(6): 1019-1031.
Michalet, X., S. Weiss and M. Jager (2006). "Single-molecule fluorescence studies of protein folding and conformational dynamics." Chem Rev 106(5): 1785-1813.
Min Jou, W., G. Haegeman, M. Ysebaert and W. Fiers (1972). "Nucleotide sequence of the gene coding for the bacteriophage MS2 coat protein." Nature 237(5350): 82-88.
Moazed, D. and H. F. Noller (1989). "Interaction of tRNA with 23S rRNA in the ribosomal A, P, and E sites." Cell 57(4): 585-597.
Moazed, D. and H. F. Noller (1989). "Intermediate states in the movement of transfer RNA in the ribosome." Nature 342(6246): 142-148.
Nissen, P., J. Hansen, N. Ban, P. B. Moore and T. A. Steitz (2000). "The structural basis of ribosome activity in peptide bond synthesis." Science 289(5481): 920-930.
Palade, G. E. (1955). "A small particulate component of the cytoplasm." J Biophys Biochem Cytol 1(1): 59-68.
Patel, S. S. and I. Donmez (2006). "Mechanisms of helicases." J Biol Chem 281(27): 18265-18268.
Petrelli, D., A. LaTeana, C. Garofalo, R. Spurio, C. L. Pon and C. O. Gualerzi (2001). "Translation initiation factor IF3: two domains, five functions, one mechanism?" EMBO J 20(16): 4560-4569.
Philippe, C., F. Eyermann, L. Benard, C. Portier, B. Ehresmann and C. Ehresmann (1993). "Ribosomal protein S15 from Escherichia coli modulates its own translation by trapping the ribosome on the mRNA initiation loading site." Proc Natl Acad Sci U S A 90(10): 4394-4398.
Philippe, C., C. Portier, M. Mougel, M. Grunberg-Manago, J. P. Ebel, B. Ehresmann and C. Ehresmann (1990). "Target site of Escherichia coli ribosomal protein S15 on its messenger RNA. Conformation and interaction with the protein." J Mol Biol 211(2): 415-426.
Portier, C., C. Philippe, L. Dondon, M. Grunberg-Manago, J. P. Ebel, B. Ehresmann and C. Ehresmann (1990). "Translational control of ribosomal protein S15." Biochim Biophys Acta 1050(1-3): 328-336.
Qu, X., J. D. Wen, L. Lancaster, H. F. Noller, C. Bustamante and I. Tinoco, Jr. (2011). "The ribosome uses two active mechanisms to unwind messenger RNA during translation." Nature 475(7354): 118-121.
Ramakrishnan, V. (2002). "Ribosome structure and the mechanism of translation." Cell 108(4): 557-572.
Rodnina, M. V., A. Savelsbergh, V. I. Katunin and W. Wintermeyer (1997). "Hydrolysis of GTP by elongation factor G drives tRNA movement on the ribosome." Nature 385(6611): 37-41.
Rosenberger, R. F. and G. Foskett (1981). "An estimate of the frequency of in vivo transcriptional errors at a nonsense codon in Escherichia coli." Mol Gen Genet 183(3): 561-563.
Schlax, P. J. and D. J. Worhunsky (2003). "Translational repression mechanisms in prokaryotes." Mol Microbiol 48(5): 1157-1169.
Schluenzen, F., A. Tocilj, R. Zarivach, J. Harms, M. Gluehmann, D. Janell, A. Bashan, H. Bartels, I. Agmon, F. Franceschi and A. Yonath (2000). "Structure of functionally activated small ribosomal subunit at 3.3 angstroms resolution." Cell 102(5): 615-623.
Schmeing, T. M., K. S. Huang, D. E. Kitchen, S. A. Strobel and T. A. Steitz (2005). "Structural insights into the roles of water and the 2'' hydroxyl of the P site tRNA in the peptidyl transferase reaction." Mol Cell 20(3): 437-448.
Schmeing, T. M., K. S. Huang, S. A. Strobel and T. A. Steitz (2005). "An induced-fit mechanism to promote peptide bond formation and exclude hydrolysis of peptidyl-tRNA." Nature 438(7067): 520-524.
Shatsky, I. N., A. V. Bakin, A. A. Bogdanov and V. D. Vasiliev (1991). "How does the mRNA pass through the ribosome?" Biochimie 73(7-8): 937-945.
Shine, J., L. Dalgarno and J. A. Hunt (1974). "Fingerprinting of eukaryotic ribosomal RNA labelled with tritiated nucleosides." Anal Biochem 59(2): 360-365.
Steitz, T. A. (2008). "A structural understanding of the dynamic ribosome machine." Nat Rev Mol Cell Biol 9(3): 242-253.
Stryer, L. and R. P. Haugland (1967). "Energy transfer: a spectroscopic ruler." Proc Natl Acad Sci U S A 58(2): 719-726.
Takyar, S., R. P. Hickerson and H. F. Noller (2005). "mRNA helicase activity of the ribosome." Cell 120(1): 49-58.
Tinoco, I., Jr. and J. D. Wen (2009). "Simulation and analysis of single-ribosome translation." Phys Biol 6(2): 025006.
Tucker, B. J. and R. R. Breaker (2005). "Riboswitches as versatile gene control elements." Curr Opin Struct Biol 15(3): 342-348.
Valle, M., A. Zavialov, J. Sengupta, U. Rawat, M. Ehrenberg and J. Frank (2003). "Locking and unlocking of ribosomal motions." Cell 114(1): 123-134.
Walter, N. G., C. Y. Huang, A. J. Manzo and M. A. Sobhy (2008). "Do-it-yourself guide: how to use the modern single-molecule toolkit." Nat Methods 5(6): 475-489.
Watson, J. D. (1963). "Involvement of RNA in the synthesis of proteins." Science 140(3562): 17-26.
Weinger, J. S., K. M. Parnell, S. Dorner, R. Green and S. A. Strobel (2004). "Substrate-assisted catalysis of peptide bond formation by the ribosome." Nat Struct Mol Biol 11(11): 1101-1106.
Wen, J. D. (2010). "解開遺傳密碼的解碼者 – 核醣體." 化學 68(4): 293-301.
Wimberly, B. T., D. E. Brodersen, W. M. Clemons, Jr., R. J. Morgan-Warren, A. P. Carter, C. Vonrhein, T. Hartsch and V. Ramakrishnan (2000). "Structure of the 30S ribosomal subunit." Nature 407(6802): 327-339.
Wu, Y. J. (2013). Using Optical Tweezers to Study the Mechanism of Structural Rearrangement in 5''UTR of rpsO mRNA. Master Thesis, National Taiwan University.
Wu, Y. J., C. H. Wu, A. Y. Yeh and J. D. Wen (2014). "Folding a stable RNA pseudoknot through rearrangement of two hairpin structures." Nucleic Acids Res 42(7): 4505-4515.
Yusupova, G. Z., M. M. Yusupov, J. H. Cate and H. F. Noller (2001). "The path of messenger RNA through the ribosome." Cell 106(2): 233-241.
Zamecnik, P. C. and E. B. Keller (1954). "Relation between phosphate energy donors and incorporation of labeled amino acids into proteins." J Biol Chem 209(1): 337-354.



QRCODE
 
 
 
 
 
                                                                                                                                                                                                                                                                                                                                                                                                               
第一頁 上一頁 下一頁 最後一頁 top