This project will continue its long term commitment of improving methods to synthesize modified tRNAs and exploit these methods to learn about tRNA function. Experiments using the well-studied E. coil glutamyl tRNA synthetase system to explore the idea that tRNA """"""""recognition"""""""" by synthetases not only involves interaction with bases, but also involves interaction with backbone functionality whose location in space is defined by the tRNA sequence. The remainder of the project involves studying the interaction of tRNA with translational apparatus. Experiments studying the interaction of modified tRNAs with elongation factor Tu are focused on understanding how the protein can bind all elongator aminoacyl tRNAs with similar affinity despite differing thermodynamic contributions of the different amino acids and tRNA bodies. A careful thermodynamic evaluation of two ternary complexes with available X-ray structures is also included. In collaboration with the Wintermeyer/Rodnina laboratory, modified tRNAs will be used to study the mechanism of the pre-translocation steps of E. coli translation. The first goal is to prepare new fluorescent tRNAs by chemical synthesis that can be used to measure rates of ribosome binding, conformational change, GTP hydrolysis, and peptide synthesis by rapid kinetic methods. Experiments using modified tRNAs will then address (1) how the correct codon match stimulates the GTPase of EF-Tu and (2) how the ribosome maintains a uniform rate of translation despite large differences in the energy of the codon-anticodon pairs. Finally, the Thermus thermophilus translation system will be developed for comparison purposes and for the potential benefit of slower rates of translation.
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