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標題: | 高通量方法定量闡明細菌調節元件的設計原理 High-Throughput Approaches Quantitatively Elucidate the Design Principles of Bacterial Regulatory Elements |
作者: | 郭學庭 Syue-Ting Kuo |
指導教授: | 周信宏 Hsin-Hung Chou |
關鍵字: | 順式調控元件,Shine-Dalgarno 序列,上位性,序列功能關係,微生物學,分子演化,細菌啟動子,轉錄調控,大規模平行報告分析,生物物理模型,合成生物學, cis-regulatory element,Shine-Dalgarno sequence,epistasis,sequence-function relationship,microbiology,molecular evolution,bacterial promoter,transcriptional regulation,massively parallel reporter assays,biophysical model,synthetic biology, |
出版年 : | 2023 |
學位: | 博士 |
摘要: | 基因表現始於基因轉錄成 mRNA,隨後 mRNA 被轉譯成蛋白質。順式調控元件(cis-regulatory element, CRE),如啟動子和核糖體結合位點(ribosomal binding site, RBS),分別決定轉錄和轉譯效率,並共同決定基因表現水平。建構功能性基因回路涉及 CRE 的耗時且反覆嘗試的優化,顯示對其序列-功能關係的理解仍不足。為了解決這個問題,在第二章中,我構建了49種RBS變體,在第三章中,我構建了412種啟動子變體,並進行了高通量實驗以測量它們的轉譯和轉錄效率。
將實驗數據與量化分析結合,我確定了三個一般性的設計原則:(1)RBS中Shine-Dalgarno序列的鳥嘌呤含量直接決定翻譯效率,儘管內源序列呈現腺嘌呤和鳥嘌呤富集的性質。(2)啟動子中的-10元件對於轉錄啟動是絕對必要的,而-35元件則不是。(3)儘管-35元件在功能上非必要,但其與RNA聚合酶的交互作用受轉錄因子調控。檢驗內源受調控的啟動子展示了一致的特性,表明這些啟動子受天擇精細地篩選。為了更深入地研究分子機制,在第二章中,我利用RNA二級結構演算法闡明了Shine-Dalgarno序列-功能關係,而在第三章中,我建構了生物物理模型來描述啟動子功能並量化RNA聚合酶和啟動子的結合能量。 我的論文強調全面的序列-功能映射的必要性,因為它提供了對 CRE 功能和演化完整的理解。這些數據集對於未來開發預測模型用以編程基因回路並識別細菌基因組中的內源 CRE 具有寶貴的價值。 Gene expression begins with transcription of genes into mRNA, followed by translation of mRNA into proteins. Cis-regulatory elements (CRE), such as promoters and ribosome binding sites (RBS), interact with the transcription and translation machinery, respectively, that jointly determine expression levels. Nevertheless, constructing a functional genetic circuit involves time-consuming and trial-and-error optimization of CRE, suggesting an insufficient understanding of their sequence-function relationship. To address this, I constructed 49 RBS variants (in Chapter 2) and 412 promoter variants (in Chapter 3) and conducted high-throughput experiments to characterize their translation and transcription efficiency, respectively. Combining my experimental data with quantitative analysis and modeling, I identified three general design principles: (1) The guanine content of the Shine-Dalgarno sequence in RBS directly determines the translation efficiency, even though endogenous sequences show an AG-rich nature. (2) The -10 element in the promoter is absolutely essential for transcription initiation, whereas the -35 element is not. (3) Although the -35 element is functionally dispensable, its interaction with RNA polymerase (RNAP) is delicately modulated by transcriptional activators. Examining endogenous regulated promoters showed a consistent pattern, suggesting that regulatory gene expression has been fine-tuned by evolution. To delve deeper into the molecular mechanisms, in Chapter 2, I utilized an RNA folding algorithm to elucidate SD sequence-function relationship, while in Chapter 3, I developed biophysical models to describe promoter function and quantify RNAP-promoter binding energy. My work underscores the necessity of global sequence-function mapping as it provides an unbiased and comprehensive understanding of CRE function and evolution. These datasets are invaluable for future development of prediction models to program genetic circuits and identify endogenous CRE in bacterial genomes. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/92418 |
DOI: | 10.6342/NTU202304478 |
全文授權: | 未授權 |
顯示於系所單位: | 生命科學系 |
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