The main focus of our proposal is the time-resolved investigation of molecular mechanisms of protein biosynthesis. Specifically, we will study the mechanism of ribosomal translocation on the picosecond to second time scale applying novel single molecule spectroscopic techniques, namely confocal optical microscopy. The ultimate goal of our proposal is to characterize molecular motions that take place in the ribosomal elongation system, 70S ribosome - aminoacyl-tRNA - peptidyl-tRNA - mRNA - elongation factor G - GTP, during the translocation cycle. Dislocation of tRNAs which takes place during ribosomal translocation will be detected by time-resolved anisotropy, and energy transfer experiments using both far- and near-field techniques. These experiments are carried out in a liquid cell, which allows to add buffer to the mica surface and models the natural ribosomal environment. The peptidyl tRNA is labeled by rhodamine B sulfonyl chloride. The aminoacyl tRNA will be labeled by 5-iodoacetamidofluorescein. According to the literature data, the distance between the peptidyl and aminoacyl tRNAs located in the 50S ribosome subunit is ~30 E. Because the fluorescein (energy donor) fluorescence spectrum overlaps the rhodamine G (energy acceptor) absorption spectrum, an efficient energy transfer occurs between these two molecules situated in the 50S. Ribosomal translocation, initiated by GTP, causes the aminoacyl tRNA displacement to the peptidyl site and the peptidyl tRNA movement out of this site, which is followed by spectral changes of fluorescent probes.
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