Single Molecule FRET Study of Ribosome Translocation

描述（由申请人提供）：A。利用单分子荧光共振能量转移（FRET）技术结合全内反射荧光显微镜（TIRFM），以Cy 3-Cy 5为供体-受体对，直接探测核糖体L11蛋白和EFG蛋白在易位过程中的相对结构变化。第一个目标是直接探测核糖体L11蛋白和EFG蛋白之间的相对结构变化在易位过程中作为时间的函数。在生物相对条件下，信息既是结构性的，又是动态的。B。我们将通过监测L11和tRNA之间的FRET更直接地研究易位。结合这一点和L11 -EFG时间依赖性FRET变化，我们将揭示EFG构象变化和实际tRNA运动的顺序或耦合。EFG构象变化与tRNA运动的耦合将有力地表明EFG是一种运动蛋白，这一点仍有争议。C.我们将把我们的研究重点扩展到涉及其他G蛋白的过程，如IF 2和EF-Tu。我们也有兴趣研究其他核糖体位置的构象变化，例如L1蛋白。

DESCRIPTION (provided by applicant): 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.