Setup of an in vitro Translation System for Single-Molecule Study

雖然關於轉譯過程的研究已經持續了五十年，但是其中仍有許多未解的部分。舉例來說，像核醣體這樣巨大且精密的聚合物是如何協調內部的構造來達到轉譯的精確性就是人們一直以來想要知道的問題。之前，生化反應的研究以及結構的資訊給了我們許多的訊息，包含轉譯過程中核醣核酸與核醣核酸、核醣核酸與蛋白質之間的交互作用以及不同狀態的核醣體。然而，像是核醣體間的異質性以及其動態的移動卻很難被觀察到。最近，許多的研究顯示我們可以利用單分子的技術來將這些片段的資訊整合成一部連環的故事。在此研究中，我們將焦點放在建立一個可以讓我們在不同條件下操縱並控制的體外轉譯系統並將其應用於單分子實驗中。因此，我們一次可以追蹤一個轉譯的過程甚至是轉譯過程中的一步。在研究的一開始，我們從大腸桿菌 Escherichia coli 上純化出轉譯過程中所需的轉譯因子以及核醣體，並且我們也證實了它們的活性。接下來，我們將這些材料組合在一起並表現冷光蛋白酶。冷光蛋白酶讓我們可以用數值定量來觀察蛋白質的表現。利用這樣的方式我們才得以建立一個最佳化的體外轉譯系統，包括緩衝液的組成、各個因子、以及溫度的調整等幾個部分。 此時，配合單分子的研究我們正在架設一組全反射螢光顯微鏡。而這兩個系統將會先用來觀察核醣體的解旋活性。Although it has been studied over half a century, the translation mechanism is still far from clear. How a large and sophisticated ribosome complex coordinates to accomplish the high translation fidelity is what people have been curious about. Biochemical studies and structure data give us lots of information including RNA-RNA, RNA-protein interactions inside the ribosome and different ribosomal states during translation. However, the heterogeneity and dynamic movements of ribosomes are hard to be observed by traditional approaches. Recently, it had been shown that single-molecule methods may help to combine these snapshots into a movie. In this study, we focus on setting up an in vitro translation system which can be manipulated and controlled under different conditions, and then applied to single molecule experiments. Therefore, we can follow the translation process one molecule and one step at a time. First, all the translation factors as well as ribosomes required for the system have been purified from Escherichia coli and confirmed to be active. These components are then put together to express luciferase protein, which allows us to quantify the expression yield. With this method, we wish to optimize the in vitro translation system in our laboratory. This system has been tuned by varying the buffer composition, the translation factors, and temperature. Now we are building a TIRF (total internal reflection fluorescence) microscope to study translation at the single-molecule level. The helicase activity of the ribosome will be studied first