Single Molecule FRET Study of Ribosome Translocation

A.使用单分子荧光共振能量转移(FRET)技术与总 内反射荧光显微镜(TIRFM)可直接探测 核糖体L11蛋白和EFG蛋白在转位过程中作为时间函数的研究 供体-受体配对。第一个目标是直接探测核糖体L11之间的相对结构变化 蛋白质和EFG蛋白在转位过程中作为时间的函数。这些信息既是结构化的，也是 在生物相对条件下是动态的。 B.我们将通过监测L11和tRNAs之间的FRET来更直接地研究易位。组合 这和L11-EFG随时间变化的FRET，我们将揭示EFG的顺序或耦合 构象变化和tRNA的实际运动。EFG构象变化与分子间的耦合作用 TRNAs的运动将强烈表明EFG是一种运动蛋白，这一点仍然存在争议。 C.我们将把研究重点扩展到涉及其他G蛋白的过程，如IF2和EF-Tu。我们 也对研究其他核糖体位置的构象变化感兴趣，例如L1蛋白。

A. Using single molecule fluorescence resonance energy transfer (FRET) technology coupled with total internal reflection fluorescence microscopy (TIRFM) to directly probe the relative structure change between ribosome L11 protein and EFG protein during translocation as a function of time using Cy3-Cy5 as the donor-acceptor pair. The first goal is to directly probe the relative structure change between ribosome L11 protein and EFG protein during translocation as a function of time. The information is both structural and dynamic under a biological relative condition. B. we will study the translocation more directly by monitoring FRET between L11 and tRNAs. Combining this and the L11 -EFG time dependent FRET changes, we will reveal the order or coupling of the EFG conformational change and the actual tRNA movements. Coupling of EFG conformation change and the tRNAs movement will be a strong indication that EFG is a motor protein, which remains controversial. C. We will extend our research thrust to processes involving other G proteins, such as IF2 and EF-Tu. We are also interested in study conformation changes of other ribosome locations, for example L1 protein.