In order to understand the accuracy of protein biosynthesis in vivo, I will investigate in vitro reactions through which ribosomes select aa-tRNAs. My past work has demonstrated that there are at least two recognition steps involved in this process, the first involving selection of an aa-tRNA-EFTu GTP complex and GTP hydrolysis, the second involving proofreading of the aa-tRNA of the complex. The title has been revised from that for the previous project period to reflect our intention to study both steps of this process. By combining quench and stopped flow techniques with product analysis and fluorescence and esr spectroscopy I expect to determine the concentrations of reactants, intermediates and starting materials during the ribosome's reaction with both the correct and incorrect substrates. These data will be used to calculate the rate and equilibrium constants necessary to predict, and understand the accuracy of this process in vivo. By studying aa-tRNA selection by ribosomes programmed with a variety of mRNAs, I expect to determine how the compositionof the codon andanticodon affect the accuracy of aa-tRNA selection and whether the context of either appreciably influenes selectivity. The work is predominantly biochemical, but techniques from both genetics and biophysics will be used. The work is relevant to health through its contribution to our knowledge of cell physiology and its disruption thorugh antibiotics or mutations. The work may prove of importance to the future use of bacteria in the production of pharmacollogically active proteins and peptides.
