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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 温進德 | zh_TW |
dc.contributor.advisor | Jin-Der Wen | en |
dc.contributor.author | 許鈺婕 | zh_TW |
dc.contributor.author | Yu-Chieh Hsu | en |
dc.date.accessioned | 2023-09-22T16:34:43Z | - |
dc.date.available | 2023-11-09 | - |
dc.date.copyright | 2023-09-22 | - |
dc.date.issued | 2023 | - |
dc.date.submitted | 2023-08-07 | - |
dc.identifier.citation | Chen, H., et al. (1994). "Determination of the optimal aligned spacing between the Shine–Dalgarno sequence and the translation initiation codon of Escherichia coli m RNAs." Nucleic acids research 22(23): 4953-4957.
Cornish, P. V. and T. Ha (2007). "A survey of single-molecule techniques in chemical biology." ACS chemical biology 2(1): 53-61. de Smit, M. H. and J. van Duin (1994). "Translational initiation on structured messengers: another role for the Shine-Dalgarno interaction." Journal of molecular biology 235(1): 173-184. Evfratov, S. A., et al. (2017). "Application of sorting and next generation sequencing to study 5΄-UTR influence on translation efficiency in Escherichia coli." Nucleic acids research 45(6): 3487-3502. Fish, K. N. (2009). "Total internal reflection fluorescence (TIRF) microscopy." Current protocols in cytometry 50(1): 12.18. 11-12.18. 13. Ha, T. (2001). "Single-molecule fluorescence resonance energy transfer." Methods 25(1): 78-86. Ivanov, I. P., et al. (2017). "Translation initiation from conserved non-AUG codons provides additional layers of regulation and coding capacity." MBio 8(3): e00844-00817. Kearse, M. G. and J. E. Wilusz (2017). "Non-AUG translation: a new start for protein synthesis in eukaryotes." Genes & development 31(17): 1717-1731. Kudla, G., et al. (2009). "Coding-sequence determinants of gene expression in Escherichia coli." science 324(5924): 255-258. Osterman, I. A., et al. (2013). "Comparison of mRNA features affecting translation initiation and reinitiation." Nucleic acids research 41(1): 474-486. Shine, J. and L. Dalgarno (1974). "The 3′-terminal sequence of Escherichia coli 16S ribosomal RNA: complementarity to nonsense triplets and ribosome binding sites." Proceedings of the National Academy of Sciences 71(4): 1342-1346. Sterk, M., et al. (2018). "Unstructured 5′-tails act through ribosome standby to override inhibitory structure at ribosome binding sites." Nucleic acids research 46(8): 4188-4199. Villegas, A. and A. M. Kropinski (2008). "An analysis of initiation codon utilization in the Domain Bacteria–concerns about the quality of bacterial genome annotation." Microbiology 154(9): 2559-2661. Wu, Y.-J., et al. (2014). "Folding a stable RNA pseudoknot through rearrangement of two hairpin structures." Nucleic acids research 42(7): 4505-4515. Lai, WJ.C., Kayedkhordeh, M., Cornell, E.V. et al. (2018). "mRNAs and lncRNAs intrinsically form secondary structures with short end-to-end distances." Nat Commun 9, 4328. 楊承翰. 2019. 以單分子螢光共振能量轉移技術研究核醣體在轉譯起始階段的搜尋機制. 臺灣大學 廖仁豪. 2020. 使用單分子螢光共振能量轉移技術研究螢光標記之核醣體亞基如何搜尋訊息核醣核酸的轉譯起始位. 臺灣大學 盧韋霖. 2020. 核醣體bS1蛋白在轉譯起始階段之功能性研究. 臺灣大學 | - |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/89894 | - |
dc.description.abstract | 在細菌的轉譯過程中,存在多個與mRNA結構相關的因素參與調控。例如,轉譯起始序列位於mRNA起始密碼子附近,提供核醣體結合位置,其結構和序列特徵影響轉譯的起始效率。轉譯起始序列包括Shine-Dalgarno序列(SD序列),它能與核醣體結合,幫助定位正確的起始密碼子。此外,細菌mRNA的非編碼區域(5' UTR和3' UTR)的一些特性亦可能具有調控轉譯的潛力,包含序列、結構和轉譯調控蛋白質(或核醣體)的結合位點。在本篇論文,我們主要利用單分子螢光共振能量轉移(smFRET)技術,觀察rpsO基因不同長度的mRNA與反義寡核苷酸 (antisense oligomers) 和30S核醣體次單元的相互作用。我們利用反義寡核苷酸與mRNA的黏合模擬30S次單元的結合,結果發現黏合的效率普遍偏低,這同時也說明了30S上其他核醣體蛋白與核醣體RNA的重要性。接下來的實驗中,觀察到標記螢光的30S核醣體次單元與三種不同長度的mRNA進行作用時,發現具有完整上下游序列的mRNA與30S核醣體次單元之間的動態結合最為明顯,這說明mRNA的長度與30S核醣體次單元的結合有高度相關的關聯性,可能和核醣體搜尋mRNA上面的轉譯起始位有關。 | zh_TW |
dc.description.abstract | Multiple factors related to mRNA structures are involved in the regulation of translation in bacteria. For instance, the translation initiation site, located near the mRNA start codon, provides a binding site for ribosomes, and its structural and sequence features influence the efficiency of translation initiation. The translation initiation site includes the Shine-Dalgarno (SD) sequence, which binds to the ribosome and helps to position the correct start codon. Translation initiation factors, such as IF1, IF2, and IF3, regulate the initiation process in bacteria. Additionally, the non-coding regions of bacterial mRNA, namely the 5' untranslated region (5' UTR) and 3' UTR, have the potential to regulate translation. These regions may contain regulatory sequences, structural elements, and binding sites for translational regulatory proteins and even ribosomes. In this study, we primarily employ the single-molecule fluorescence resonance energy transfer (smFRET) technique to observe the interaction between antisense oligomers, as well as the 30S ribosomal subunit, and the mRNA of the rpsO gene with varying lengths. We anneal antisense oligomers to mRNA to mimic the binding of the 30S ribosomal subunit. The results showed that the efficiency is generally low, suggesting the importance of ribosomal proteins and ribosomal RNA on the 30S subunit. However, observations of fluorescence-labeled 30S subunits interacting with mRNAs of different lengths indicated that mRNAs containing the complete downstream coding sequence exhibited the most dynamic binding with the 30S subunit. These preliminary data suggest a significant correlation between the length of mRNA and the 30S ribosomal subunit, which may be related to the searching mechanism of 30S subunits for the translation initiation site. | en |
dc.description.provenance | Submitted by admin ntu (admin@lib.ntu.edu.tw) on 2023-09-22T16:34:43Z No. of bitstreams: 0 | en |
dc.description.provenance | Made available in DSpace on 2023-09-22T16:34:43Z (GMT). No. of bitstreams: 0 | en |
dc.description.tableofcontents | 目錄
致謝 II 摘要 III ABSTRACT IV 第一章 緒論 - 1 - 1.1轉譯起始 - 1 - 1.2單分子技術與螢光共振能量轉移 - 1 - 1.3 RPSO - 2 - 1.4 MRNA結構序列 - 2 - 1.5動機 - 3 - 第二章 實驗材料 - 5 - 2.1 PLASMIDS - 5 - 2.2 OLIGO - 5 - 2.3 CELL LINES - 6 - 2.4溶液 - 7 - 2.5酵素 - 10 - 2.6藥品 - 12 - 2.7 KIT - 13 - 第三章 實驗方法 - 14 - 3.1 POLYMERASE CHAIN REACTION(PCR) - 14 - 3.2 載體構築 - 17 - 3.3 MRNA合成 - 17 - 3.4 EMSA實驗 - 18 - 3.5 30S核醣體次單元之純化 - 19 - 3.6 30S核醣體次單元標定螢光 - 21 - 3.7 單分子螢光共振能量轉移實驗 (SMFRET) - 22 - 3.8 數據分析 - 25 - 第四章 結果 - 26 - 4.1 30S核醣體次單元純化、活性測定與螢光標定 - 26 - 4.2 EMSA實驗檢測DNA探針與MRNA的黏合 - 27 - 4.3 SMFRET實驗檢測DNA探針與MRNA的黏合 - 28 - 4.4 MRNA FRET值分布 - 30 - 4.5 MRNA與30SWT結合 - 31 - 第五章 討論 - 32 - 5.1 MRNA長度愈長會使DNA探針黏合更佳 - 32 - 5.2 黏合結構位置影響效率 - 32 - 5.3 MRNA結構對修飾CY3 DNA探針配對效能的影響 - 33 - 5.4 RPSOUTR有較好黏合比例與其構形有關聯性 - 33 - 5.5 黏合位暴露與否與效率相關 - 34 - 5.6 RPSOFULL REALTIME與PRE-ANNEAL結果差異 - 35 - 參考文獻 - 36 - 圖表 - 38 - 圖1.T7SP6Vec質體構築示意圖與部分序列示意圖 - 39 - 圖2. 單分子螢光共振能量轉移實驗 (smFRET) - 40 - 圖3. 純化之30S核醣體次單元進行SDS-PAGE 結果 - 41 - 圖4. 以F+18 mRNA測試 30S結合的活性 - 42 - 圖5. 以mS1L mRNA測試30S結合的活性 - 45 - 圖6. 以RPSOutr mRNA測定30SS6-ATTO結合活性 - 46 - 圖7. RPSOfull、RPSOutr、RPSOrbs44序列及結構示意圖 - 48 - 圖8. (T7) RPSOfull、RPSOutr、RPSOrbs44序列及結構示意圖 - 49 - 圖9. Cy3-5’端探針與RPSO系列mRNA黏合實驗 - 50 - 圖10. Cy3-RBS探針與RPSO系列mRNA黏合實驗 - 51 - 圖11. smFRET實驗Pre-anneal及Real time示意圖 - 52 - 圖12. smFRET實驗分析示意圖 - 53 - 圖13. Cy3-5’端探針Pre-anneal狀態下螢光強度族群分布圖 - 54 - 圖14. Cy3-5’端探針Real time與Pre-anneal螢光強度分布疊合圖 - 55 - 圖15. Cy3-5’端探針Pre-anneal與Realtime黏合效率差異原因示意圖 - 56 - 圖16. Cy3-RBS探針Pre-anneal狀態下螢光強度族群分布圖 - 58 - 圖17. Cy3-RBS探針Real time與Pre-anneal螢光強度分布疊合圖 - 59 - 圖18. Cy3-RBS探針Pre-anneal與Realtime黏合效率差異原因示意圖 - 60 - 圖19. mRNA與Cy3-DNA探針黏合之smFRET實驗 - 62 - 圖20. RPSO mRNA FRET分布與動態 - 63 - 圖21. 30S對RPSO mRNA FRET的影響 - 65 - 表1. 論文中所使用的質體存放位置 - 67 - 表2. Cy3-5’端探針EMSA 實驗數據 - 68 - 表3. Cy3-RBS探針EMSA 實驗數據 - 68 - | - |
dc.language.iso | zh_TW | - |
dc.title | 以單分子螢光共振能量轉移觀測mRNA的長度對反義寡核苷酸以及30S核醣體次單元的交互作用影響 | zh_TW |
dc.title | Effects of mRNA Lengths on the Interaction with Antisense Oligomers and 30S Ribosomal Subunits by Single-Molecule FRET | en |
dc.type | Thesis | - |
dc.date.schoolyear | 111-2 | - |
dc.description.degree | 碩士 | - |
dc.contributor.oralexamcommittee | 李弘文;李以仁 | zh_TW |
dc.contributor.oralexamcommittee | Hung-Wen Li;I-Ren Li | en |
dc.subject.keyword | 轉譯起始,rpsO,非編碼區域,30S次單元,核醣體,單分子螢光共振能量轉移, | zh_TW |
dc.subject.keyword | Translation initiation,rpsO,non-coding regions,30S subunit,ribosome,Single-Molecule Fluorescence Resonance Energy Transfer, | en |
dc.relation.page | 68 | - |
dc.identifier.doi | 10.6342/NTU202303183 | - |
dc.rights.note | 未授權 | - |
dc.date.accepted | 2023-08-09 | - |
dc.contributor.author-college | 生命科學院 | - |
dc.contributor.author-dept | 分子與細胞生物學研究所 | - |
顯示於系所單位: | 分子與細胞生物學研究所 |
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