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研究生(外文):Yi-Ju Wu
論文名稱(外文):Using Optical Tweezers to Study the Mechanism of Structural Rearrangement in 5’UTR of rpsO mRNA
指導教授(外文):Jin-Der Wen
外文關鍵詞:translational regulationrpsO mRNAstructural rearrangementsingle-moleculeoptical tweezers
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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
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
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