Single Molecule FRET Study of Ribosome Translocation

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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.