指導教授：温進德臺灣大學：分子與細胞生物學研究所陳郁庭Chen, Yu-TingYu-TingChen2014-11-252018-07-062014-11-252018-07-062014http://ntur.lib.ntu.edu.tw//handle/246246/261097人類端粒酶是由451個鹼基的核醣核酸、一種特定的反轉錄酶(human telomerase reverse transcriptase)以及其他相關的蛋白質所組成。在人類端粒酶核醣核酸的5’ 包含了與端粒形成有關的核醣核酸模板(RNA template)，以及和端粒酶活性有關的高度保存(highly conserved)偽結結構(pseudoknot)。 此高度保存的偽結結構包括了2個類髮夾(hairpin)的結構，2個類髮夾互相作用在結構中形成了大凹槽(major-groove)與小凹槽(minor-groove)，在2個凹槽中有5對三重鹼基對(base-triple)，過去利用偽結突變構造 (hTR DU177)的研究指出，偽結結構之穩定度會受到內部三重鹼基對的影響，當三重鹼基對形成，其穩定性相對提高，但至今對於偽結結構是如何折疊而成的仍沒有一定論。 本研究即是藉由單分子技術，以雷射光鉗(optical tweezers)對hTR DU177施加外力，觀察外力與打開長度之間的關係，發現在打開結構的過程中大部分的是先打開其中一個類髮夾結構，只有在少數情況下會打開整個偽結結構，之後再經過更進一步的實驗後，我們推論形成偽結結構的途徑有2種，絕大多數的情形下偽結是以第一類髮夾結構(hairpin 1)存在居多，要形成完整的偽結結構須花較長時間，但是一旦第二類髮夾結構(hairpin 2) 比第一類髮夾結構先形成，則可迅速形成帶有三重鹼基對的偽結結構。Human telomerase is composed of a 451 nt RNA (hTR) and several proteins, including a specialized reverse transcriptase (hTERT). The 5’ domain of hTR contains a RNA template for telomere synthesis and a highly conserved pseudoknot structure crucial for telomerase activity. The highly conserved pseudoknot structure has two hairpin (stem-loop) structures. The interaction of two hairpin structures formed a major-groove and a minor-groove, as well as five triple-base pairs between them. Previous experiments have shown that the stability of the pseudokont derived from hTR (hTR DU177) was related to the base triples. However, the mechanism of pseudoknot folding is not well understood. In this study, we used optical tweezers to measure the unfolding force of a series of hTR DU177-related structures and analyzed the relationship between the force and the extension. Instead of being completely unfolded in one step, we found that only one of the two hairpins within the pseudoknot was formed and unfolded during most of the recordings. Based on further experiments, we propose that the pseudoknot is formed by two alternative pathways: Formation of the two hairpin structures kinetically competes to each other. DU177 often tends to form the stable hairpin 1 rapidly, but proceeds to fold into the complete pseudoknot in a much slower fashion. On the other hand, the RNA occasionally folds into an intact pseudoknot structure quickly via the less preferred hairpin 2 pathway.謝誌 i 摘要 ii Abstract iii Chapter 1　Introduction 1 1.1 Human Telomerase RNA 1 1.1.1 The Pseudoknot Structure of the 5’ Human Telomerase RNA (DU177) 1 1.1.2 The Relationship Between Structural Stability and Protonation 2 1.1.3 DU177 and Bimolecular Pseudoknot 2 1.2 Application of Optical tweezers 2 1.3 Specific Aims 4 1.3.1 The Effect of pH to Base triples 4 1.3.2 The Role of Base triples in Structural Dynamics 4 1.3.3 The Possible Mechanism for DU177 Pseudoknot Folding 4 Chapter 2　Materials and Methods 6 2.1 Materials 6 2.1.1 Bacterial Strains and Plasmid 6 2.1.2 DNA Primers and RNA Oligos 6 2.1.3 Chemicals 8 2.1.4 Enzymes 9 2.1.5 Kits 9 2.1.6 Buffers 10 2.2 Methods 10 2.2.1 Plasmid Constructions 10 2.2.2 In vitro Transcription 11 2.2.3 PCR for Handles 12 2.2.4 Modification of Handles 12 2.2.5 Annealing of DNA Handles and RNA 13 2.2.6 RNA Hairpin and Single-Stranded RNA Oligo Hybrid Construction 13 2.2.7 Mini-tweezers Experiments 14 Chapter 3 Results 16 3.1 The pH-dependent behaviors of the DU177 Mutant 16 3.2 The Effect of Intermolecular Interactions in DU177 17 3.3 The Folding Mechanism of DU177 19 3.3.1 Force-ramp 19 3.3.2 Narrowing down the range of pulling force 21 3.3.3 Force-drop 22 3.3.4 Bimolecular Pseudoknots 23 3.3.5 Troubleshooting of uu-hp1 and WLC 25 Chapter 4 Discussion 27 4.1　The pH-dependence of DU177 Mutant 27 4.2　The Effect of Intermolecular Interactions in DU177 27 4.3　The Folding Mechanism of DU177: The Competition Between Hairpin 1 and Hairpin 2 28 4.3.1　The study of DU177 and its mutant 28 4.3.2　The study of bimolecular pseudoknots 29 4.4　The Mismatching of uu-hp1 and its WLC 30 4.5　Perspectives 31 4.5.1　The effect of additional guanine nucleotides in 5’ unbinding nucleotides of ssRNA 31 4.5.2　The observation of conformational dynamics by smFRET 31 References 32 Figure 1. hTR DU177 sequence and the corresponding position of DNA handles 36 Figure 2. Experimental setup of optical tweezers. 37 Figure 3. The mutant 175/176T. 39 Figure 4. The mutant uu-175/176T. 41 Figure 5. The comparison of uu-DU177 and uu-175/176T in pH 6.8 buffer. 43 Figure 6. The comparison of uu-DU177 and uu-175/176T in pH 7.6 buffer. 45 Figure 7. Previous results of the effect of intermolecular interaction in DU177 (Chang 2012). 46 Figure 8. The Effect of intermolecular interactions in DU177. 47 Figure 9. The comparison of uu-DU177, hp2 and uu-hp1. 49 Figure 10. The more stable the hairpin 2 is, the more pseudoknot forms. 50 Figure 11. Increasing the refolding time did not increase the formation of pseudoknot of uu-DU177. 51 Figure 12. The pseudoknot conformation occurred occasionally even under the stringent condition. 52 Figure 13. The percentage of various conformations under the stringent condition. 53 Figure 14. To confirm the folding conformation by force-drop experiments. 54 Figure 15. The comparison between uu-DU177 and two hairpin constructs in force-drop experiments. 56 Figure 16. The equilibrium of the folding and unfolding of uu-hp1/hTR 3’ssRNA. 57 Figure 17. Bimolecular complexes of hp2. 58 Figure 18. The fraction of refolding was increased by 5 s refolding time. 59 Figure 19. The force-drop data of hp2/5’ssRNA-14. 60 Figure 20. Dwell time differences between hp2/5’ssRNA-14 and hp2. 61 Figure 21. Two possible mechanisms of forming the pseudoknot. 62 Figure 22. The mismatching of uu-hp1 and its WLC (cc-DU177). 63 Figure 23. The mismatching of uu-hp1 and its WLC (uu-ohp1). 64 Figure 24. The mismatching of uu-hp1 and its WLC (bimolecular complexes). 651991884 bytesapplication/pdf論文公開時間：2017/09/02論文使用權限：同意有償授權(權利金給回饋學校)偽結結構髮夾結構三重鹼基對雷射光鉗單分子thesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/261097/1/ntu-103-R01b43017-1.pdf