在細胞中，信使核醣核酸 (mRNA)的轉譯起始作用能夠藉由許多方式來調控，其中常見的一種方式是藉由改變核醣核酸的結構來調控轉譯作用。大腸桿菌的核醣體蛋白S15，是由rpsO基因轉譯產生，S15會與帶有其遺傳訊息的核醣核酸的五端未轉錄區域作用藉此調控S15本身的生合成。當S15在細胞中的濃度過高時，S15會與其五端未轉錄區域的核醣核酸結合使得核醣體無法進行轉譯作用，進而抑制S15的生成，降低原先在細胞中過高的S15濃度。而rpsO基因五端未轉錄區域的核醣核酸能夠折疊成假結 (pseudoknot)或者是雙髮夾 (double-hairpin)結構，這兩種結構在溶液中的穩定度相似，但是SD (Shine-Dalgarno)序列只有在假結結構時會暴露出來且與核醣體結合，進而解開假結結構起始轉譯作用。假結結構對於調控轉譯作用的功能在過去的研究中已經有相當的瞭解，但是雙髮夾的作用至今仍然不明。
因此我們想要藉由光鉗 (optical tweezers)這項技術去探究rpsO基因五端未轉錄區域在結構上的轉換機制，光鉗能夠觀察單一個核醣核酸分子的構型改變，我們發現雙髮夾能夠重組構型促進假結形成，也發現了穩定性不同的假結結構以及其他三級結構。從我們的實驗結果，我們推論雙髮夾是形成假結結構過程中重要的中間物之一。

Translation initiation of mRNA can be regulated through different ways in the cell. One of the common mechanisms is to modulate the structural elements of mRNA. Escherichia coli ribosomal protein S15 (encoded by the rpsO gene) regulates its own biosynthesis by interacting with the 5’ untranslated region (5’UTR) of its cognate mRNA. When S15 is synthesized in excess in the cell, the protein represses translation via binding to the 5’UTR of its mRNA and blocks the ribosome from accessing the initiation site. The 5’UTR region of rpsO mRNA can fold into either a pseudoknot or a double-hairpin structure, but only the former can bind the ribosome and S15. The pseudoknot and double-hairpin structures exist in equilibrium in solution. While the pseudoknot form has been dissected extensively in previous studies, the function of the double-hairpin is still unknown.
In this study, we manage to characterize the structural dynamics of the 5’UTR of the rpsO mRNA by using optical tweezers. This technique allows us to observe conformational change of single RNA molecules in real time. Our results show that the double-hairpin structure can be rearranged to the pseudoknot conformation. We also observed some structures other than the typical pseudoknot and double-hairpin; additionally, those structures are a complicated mixture. In conclusion, we suggest that the double-hairpin structure may be a necessary and key intermediate in the folding pathway to the pseudoknot and that some other tertiary structures can form from the same sequence, though their functions remain elusive.

口試委員審定書 i
致謝 ii
中文摘要 iii
ABSTRACT iv
CONTENTS v
LIST OF FIGURES viii
LIST OF TABLES x
Chapter 1 Introduction 1
1.1 The E. coli ribosomal protein S15 1
1.2 Translational autoregulation of S15 2
1.3 Application of optical tweezers 3
1.4 Specific aim 4
Chapter 2 Materials and methods 5
2.1 Materials 5
2.1.1 Bacterial Strains 5
2.1.2 Plasmid 5
2.1.3 Oligomers and Primers 5
2.1.4 Enzymes 9
2.1.5 Chemicals 9
2.1.6 Kits 11
2.1.7 Buffers 11
2.2 Methods 12
2.2.1 Construction of plasmids 12
2.2.2 In vitro transcription 12
2.2.3 PCR for handles 12
2.2.4 Modification of handles 13
2.2.5 Annealing reaction of DNA handles and RNA 14
2.2.6 Optical tweezers experiments 14
Chapter 3 Results 17
3.1 Preparation of RNA constructs 17
3.2 Mg2+ ions stabilize the pseudoknot structure of S15WT RNA 17
3.3 Characterization of WT force-extension patterns 18
3.3.1 HT 18
3.3.2 2CT and PK 19
3.3.3 2T 20
3.4 Further characterization of 2T pattern 21
3.5 Double-hairpin assists the formation of pseudoknot 23
3.6 The mechanism for the rearrangement of double-hairpin into pseudoknot. 24
3.7 Molecular dissection of the pseudoknot structure 25
Chapter 4 Discussions 27
4.1 Mg2+ Ions stabilize Tertiary Structures 27
4.2 Partial 2Ts resulted from unidentified tertiary structures. 27
4.3 Double-hairpin assists the formation of pseudoknot 28
4.4 Moderately unstable double-hairpins can rearrange into pseudoknots 29
4.5 Structural stability of 2T, 2CT and PK. 30
4.6 Double-hairpin might be an intermediate on the folding pathway of pseudoknot. 31
4.7 Perspectives. 31
References 33

Abbondanzieri, E. A., W. J. Greenleaf, J. W. Shaevitz, R. Landick and S. M. Block (2005). "Direct observation of base-pair stepping by RNA polymerase." Nature 438(7067): 460-465.

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.

Ashkin, A., J. M. Dziedzic, J. E. Bjorkholm and S. Chu (1986). "Observation of a single-beam gradient force optical trap for dielectric particles." Opt Lett 11(5): 288.

Benard, L., N. Mathy, M. Grunberg-Manago, B. Ehresmann, C. Ehresmann and C. Portier (1998). "Identification in a pseudoknot of a U.G motif essential for the regulation of the expression of ribosomal protein S15." Proc Natl Acad Sci U S A 95(5): 2564-2567.

Block, S. M., L. S. Goldstein and B. J. Schnapp (1990). "Bead movement by single kinesin molecules studied with optical tweezers." Nature 348(6299): 348-352.

Bustamante, C., Y. R. Chemla, N. R. Forde and D. Izhaky (2004). "Mechanical processes in biochemistry." Annu Rev Biochem 73: 705-748.

Chen, G., J. D. Wen and I. Tinoco, Jr. (2007). "Single-molecule mechanical unfolding and folding of a pseudoknot in human telomerase RNA." RNA 13(12): 2175-2188.

Dumont, S., W. Cheng, V. Serebrov, R. K. Beran, I. Tinoco, Jr., A. M. Pyle and C. Bustamante (2006). "RNA translocation and unwinding mechanism of HCV NS3 helicase and its coordination by ATP." Nature 439(7072): 105-108.

Greenleaf, W. J., K. L. Frieda, D. A. Foster, M. T. Woodside and S. M. Block (2008). "Direct observation of hierarchical folding in single riboswitch aptamers." Science 319(5863): 630-633.

Hansen, T. M., S. N. Reihani, L. B. Oddershede and M. A. Sorensen (2007). "Correlation between mechanical strength of messenger RNA pseudoknots and ribosomal frameshifting." Proc Natl Acad Sci U S A 104(14): 5830-5835.

Held, W. A., B. Ballou, S. Mizushima and M. Nomura (1974). "Assembly mapping of 30 S ribosomal proteins from Escherichia coli. Further studies." J Biol Chem 249(10): 3103-3111.

Joo, C., H. Balci, Y. Ishitsuka, C. Buranachai and T. Ha (2008). "Advances in single-molecule fluorescence methods for molecular biology." Annual Review of Biochemistry 77: 51-76.

Kerssemakers, J. W., E. L. Munteanu, L. Laan, T. L. Noetzel, M. E. Janson and M. Dogterom (2006). "Assembly dynamics of microtubules at molecular resolution." Nature 442(7103): 709-712.

Lang, M. J., C. L. Asbury, J. W. Shaevitz and S. M. Block (2002). "An automated two-dimensional optical force clamp for single molecule studies." Biophys J 83(1): 491-501.

Li, P. T., C. Bustamante and I. Tinoco, Jr. (2007). "Real-time control of the energy landscape by force directs the folding of RNA molecules." Proc Natl Acad Sci U S A 104(17): 7039-7044.

Li, P. T. and I. Tinoco, Jr. (2009). "Mechanical unfolding of two DIS RNA kissing complexes from HIV-1." J Mol Biol 386(5): 1343-1356.

Liphardt, J., S. Dumont, S. B. Smith, I. Tinoco, Jr. and C. Bustamante (2002). "Equilibrium information from nonequilibrium measurements in an experimental test of Jarzynski''s equality." Science 296(5574): 1832-1835.

Mammen, M., K. Helmerson, R. Kishore, S. K. Choi, W. D. Phillips and G. M. Whitesides (1996). "Optically controlled collisions of biological objects to evaluate potent polyvalent inhibitors of virus-cell adhesion." Chem Biol 3(9): 757-763.

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.

Mikhailenko, S. V., Y. Oguchi and S. Ishiwata (2010). "Insights into the mechanisms of myosin and kinesin molecular motors from the single-molecule unbinding force measurements." J R Soc Interface 7 Suppl 3: S295-306.

Neuman, K. C. and S. M. Block (2004). "Optical trapping." Rev Sci Instrum 75(9): 2787-2809.

Nikulin, A., A. Serganov, E. Ennifar, S. Tishchenko, N. Nevskaya, W. Shepard, C. Portier, M. Garber, B. Ehresmann, C. Ehresmann, S. Nikonov and P. Dumas (2000). "Crystal structure of the S15-rRNA complex." Nat Struct Biol 7(4): 273-277.

Onoa, B., S. Dumont, J. Liphardt, S. B. Smith, I. Tinoco, Jr. and C. Bustamante (2003). "Identifying kinetic barriers to mechanical unfolding of the T. thermophila ribozyme." Science 299(5614): 1892-1895.

Philippe, C., L. Benard, C. Portier, E. Westhof, B. Ehresmann and C. Ehresmann (1995). "Molecular dissection of the pseudoknot governing the translational regulation of Escherichia coli ribosomal protein S15." Nucleic Acids Res 23(1): 18-28.

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., L. Dondon and M. Grunberg-Manago (1990). "Translational autocontrol of the Escherichia coli ribosomal protein S15." J Mol Biol 211(2): 407-414.

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.

Rice, S. E., T. J. Purcell and J. A. Spudich (2003). "Building and using optical traps to study properties of molecular motors." Methods Enzymol 361: 112-133.

Rittie, L. and B. Perbal (2008). "Enzymes used in molecular biology: a useful guide." J Cell Commun Signal 2(1-2): 25-45.

Sambrook, J. and D. W. Russell (2006). "Labeling 3'' Termini of Double-stranded DNA with Bacteriophage T4 DNA Polymerase." CSH Protoc 2006(1).

Schlax, P. J. and D. J. Worhunsky (2003). "Translational repression mechanisms in prokaryotes." Mol Microbiol 48(5): 1157-1169.

Serganov, A., E. Ennifar, C. Portier, B. Ehresmann and C. Ehresmann (2002). "Do mRNA and rRNA binding sites of E.coli ribosomal protein S15 share common structural determinants?" J Mol Biol 320(5): 963-978.

Smith, D. E., S. J. Tans, S. B. Smith, S. Grimes, D. L. Anderson and C. Bustamante (2001). "The bacteriophage straight phi29 portal motor can package DNA against a large internal force." Nature 413(6857): 748-752.


Soto, A. M., V. Misra and D. E. Draper (2007). "Tertiary structure of an RNA pseudoknot is stabilized by "diffuse" Mg2+ ions." Biochemistry 46(11): 2973-2983.

Stryer, L. and R. P. Haugland (1967). "Energy transfer: a spectroscopic ruler." Proc Natl Acad Sci U S A 58(2): 719-726.

Svoboda, K. and S. M. Block (1994). "Force and velocity measured for single kinesin molecules." Cell 77(5): 773-784.

Svoboda, K., C. F. Schmidt, B. J. Schnapp and S. M. Block (1993). "Direct observation of kinesin stepping by optical trapping interferometry." Nature 365(6448): 721-727.

Tucker, B. J. and R. R. Breaker (2005). "Riboswitches as versatile gene control elements." Curr Opin Struct Biol 15(3): 342-348.

Veigel, C. and C. F. Schmidt (2011). "Moving into the cell: single-molecule studies of molecular motors in complex environments." Nat Rev Mol Cell Biol 12(3): 163-176.

Wen, J. D., L. Lancaster, C. Hodges, A. C. Zeri, S. H. Yoshimura, H. F. Noller, C. Bustamante and I. Tinoco (2008). "Following translation by single ribosomes one codon at a time." Nature 452(7187): 598-603.

Wen, J. D., M. Manosas, P. T. Li, S. B. Smith, C. Bustamante, F. Ritort and I. Tinoco, Jr. (2007). "Force unfolding kinetics of RNA using optical tweezers. I. Effects of experimental variables on measured results." Biophys J 92(9): 2996-3009.

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.