- 學位論文
以電子顯微鏡解析酵母菌 RNA 聚合酶 II 與 Spt4-Spt5 複合體之三維結構
Three Dimensional Electron Microscopy Reconstruction of Yeast RNA Polymerase II with Spt4-Spt5 complex
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摘要
控制基因表現對於細胞存活是很重要的步驟，原核生物與真核生物都具有 RNA polymerase 以調控轉錄作用。轉錄分為起始、延長，及終止三個不同步驟，早期研究則發現轉錄延長階段為 mRNA 合成的關鍵步驟。 真核細胞之細胞核內至少有三種不同 RNA polymerase，其中RNA Pol I 主要負責合成 rRNA，RNA Pol II 負責合成 mRNA，而RNA Pol III 負責合成 tRNA。本論文將研究重點指向酵母菌 RNA Pol II 與 Spt4-Spt5 複合體。早期對 Spt4-Spt5 複合體的研究認為，它在酵母菌轉錄初始階段扮演轉錄因子角色，並調控核染色質結構。然而，近來此複合體已漸漸被證實為細胞內轉錄延長調控因子。 本篇論文中，利用電子顯微鏡的技術輔以單粒子影像分析與三維重建，決定 RNA Pol II 與 Spt4-Spt5 複合體之結構。本論文採取三種策略來增加重建結果的可信度與精確度。首先，以 reference-free 的方式重建 RNA Pol II 與 Spt4-Spt5 複合體結構，克服使用初始模型進行重建所造成的結構失真。接著進行胞外轉錄實驗證實我們所重建的結構具有生理活性，之後將使用奈米金標定的技術與 random conical tilt 的方式，增加定位 Spt4-Spt5 複合體的準確度。
並列摘要
The control of gene expression is essential for cell survival and it is commonly achieved by regulating transcription with different forms of RNA polymerase in both prokaryotes and eukaryotes. Transcription can be mainly divided into initiation, elongation and termination phases and previous studies determined elongation phase as rate-limiting step for proper mRNA production. At least three different RNA polymerase has been identified in eukaryotic cells, including RNA polymerase I for ribosome, RNA polymerase II (RNA Pol II) for mRNA and RNA polymerase III for transfer-RNA. In this study, we focus on RNA Pol II coupled with Spt4-Spt5 complex in Saccharomyces cerevisiae. Initial studies of Spt4-Spt5 complex in Saccharomyces cerevisiae suggested that they act as transcription factors that modify chromatin structure in transcription initiation. More recently, however, this complex has been generally believed to work as an elongation regulator in vivo. Here, we present the structure of the yeast RNA Pol II with Spt4-Spt5 complex determined by electron microscopy followed by single-particle analysis and three-dimensional reconstruction. Three methods have been adapted to increase the reliability and accuracy of our final structural model: First, we use reference-free methods to reconstruct the structure of RNA Pol II and Spt4-Spt5 complex which overcomes the distortion caused by reference bias using initial model-based method, and secondly, the complex have been assayed by in vitro transcription to show their biological activity. In the future, we will use gold particle labeling and random conical tilt method to increase the accuracy in identifing the position of Spt4-Spt5 in the complex.
參考文獻
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