2019-01-012024-05-14https://scholars.lib.ntu.edu.tw/handle/123456789/660178摘要：細菌產生蛋白質的第一步為核糖體亞基在mRNA的核糖體結合位點 (RBS) 上聚成完整的核糖體，此起始步驟對蛋白生成的速率有極大的影響，其分子機制在演化上具高度保守性。前人發展出預測RBS轉譯起始效率的電腦模式，儘管這類研究曾以少量實驗數據佐證，我發現電腦模式無法預估mRNA二級結構對轉譯起始作用的影響，此結果凸顯現存模式的缺失，亦反映學界對RNA在細胞內的折疊狀況以及核糖體與mRNA的互動所知有限。為增進模式預測的精準度及對蛋白轉譯的基礎知識，我研發一套結合序列建庫、螢光細胞分選、及次世代定序的高通量技術，可精準測定百萬組mRNA變異序列對轉譯起始的影響，此技術之通量超越現行核糖體印跡測序法 (ribosome profiling) 達370倍。我將以上述實驗建構mRNA基因型-表型關係的完整圖譜，對照前人電腦模式的預測，找出預測失準的案例及原因，進而改善模式預測的準確性。本研究不但會產生具不同轉譯效能的RBS模組，改進RNA二級結構預測的準確度，還將發展出更精準並可用於合成生物學的RBS功能預測軟體。<br> Abstract: Protein translation in bacteria begins with the assembly of a mature ribosome at the ribosome binding site (RBS) on mRNA. The molecular mechanisms underlying translation initiation are highly conserved, and computational models based on well-characterized thermodynamic variables have been built to assist designing RBSs of various translation efficiencies for synthetic biology. Although computational studies claimed the precision of in silico prediction, I showed the poor performance of these models by experimentally synthesizing RBS sequence variants with distinct mRNA secondary structure. This preliminary trial not only questions the accuracy of computational prediction but also hints biophysical factors missing from the current picture of translation initiation. To deepen our understanding of this fundamental process and improve model prediction, I have developed a high-throughput experiment capable of quantifying the translation efficiency of millions of RBS variants. This novel approach, combining large sequence library generation, fluorescence-activated cell sorting, and next generation sequencing will yield a comprehensive genotype-phenotype map of RBS unachievable by either conventional methods or ribosome profiling. Comparison between genotype-phenotype maps generated by experiments and computational predictions will identify mRNA secondary structure whose properties are omitted or misinterpreted by current knowledge. Incorporation of these neglected variables should improve the reliability of computational models for biotechnological application.高通量轉譯起始作用RNA二級結構電腦模式序列空間high-throughputtranslation initiationRNA secondary structurecomputational modelsequence space