Molecular Mechanism of Translation

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DESCRIPTION (provided by applicant): Protein synthesis is a fundamental process in all living organisms. During the elongation phase of protein synthesis, the ribosome accurately selects the aminoacyl-tRNA corresponding to the mRNA codon. The long-term objective of this proposal is to study the mechanism of tRNA selection by the ribosome. Structural studies revealed that the ribosome recognizes the shape of the codon-anticodon duplex during tRNA selection. However, the relative contributions of these contacts toward tRNA selection and ribosomal accuracy are not known. Therefore, the major goal of this proposal is to determine the contribution of specific contacts between the ribosome and the mRNA-tRNA complex in tRNA selection. We will also study how miscoding antibiotics affect the process of tRNA selection. We have developed a new, fluorescence-based, pre-steady state kinetic assay for studying tRNA selection. This powerful method will permit us to determine which contacts within the ribosome are most important for tRNA selection. In addition, this proposal will use quench-flow methods, site-directed mutagenesis of critical residues, and biochemical assays to study the mechanism of tRNA selection by the ribosome. These experiments will provide information about the dynamics of tRNA selection that cannot be easily acquired by X-ray crystallography. Ribosomes are the target for inactivation by several classes of antibiotics. Antibiotics such as paromomycin, streptomycin, tetracycline, and neomycin affect tRNA selection by the ribosome. Streptomycin is used to treat tuberculosis and tetracycline is used to treat anthrax infections. Antibiotic-resistant strains of bacteria are on the rise, causing a crisis in the management and treatment of these infections throughout the world. Understanding the mechanism of translation will provide insights for developing more effective antibiotics that target the ribosome of these drug-resistant strains of bacteria and infectious bioterrorism agents. Furthermore, this proposal will have a broad impact on fields such as RNA catalysis, origin of life, RNA structure and function, mechanism of eukaryotic protein synthesis, cancer, and neuroscience.