Transfer RNAs (tRNAs) interact with aminoacyl tRNA synthetases, elongation factor Tu (EF-Tu), and ribosomes to participate in the decoding of genetic information. Studies of the molecular interactions between tRNAs and synthetases have led to insights into how synthetases distinguish tRNAs on the basis of different sets of nucleotides that are unique to each tRNA for specific aminoacylation. Little is known about the interactions between tRNAs and EF-Tu, and between tRNAs and ribosomes, although the general belief is that these interactions depend on nucleotides that are common to most tRNAs. The sequences of tRNAs contain 15 conserved and 17 semi-conserved nucleotides that are common to most tRNAs. These nucleotides establish a network of hydrogen bonding interactions (the tertiary interactions) that collectively contribute to the folded, L-shaped three-dimensional tRNA structure. Preliminary studies show that substitutions at some of the conserved and semi-conserved nucleotides result in non-functional tRNAs in E. coli. The molecular basis for such effects is not understood, because substitutions may disrupt the tertiary interactions, or they may eliminate determinants that are recognized by EF-Tu or ribosomes. Among the latter possibilities, the goal is to determine those substitutions that affect the interaction with EF-Tu. The emphasis on EF-Tu is because this protein has a well defined crystal structure that can provide the framework for understanding its interaction with tRNAs. This proposal has two principle objectives. First, we will establish functional and non-functional nucleotides at each of the tertiary interactions that define the tRNA structure. Nucleotide substitutions will be systematically introduced and tested for their effects on the structure and function of a specific tRNA. Based on the crystal structure of yeast tRNAPhe, we will determine whether we can predict the types of nucleotide substitutions that can maintain the structure. Second, for those substitutions that maintain the structure but not the function, we will determine whether they eliminate the ability of tRNA to interact with EF-Tu. When substitutions that result in defective interaction with EF-Tu are identified, we will take a genetic approach to look for mutations in EF-Tu that compensate for the defect. These EF-Tu mutants, referred to as second-site revertants, will harbor mutations at sites that are critical for tRNA interaction. The proposed studies will not only advance our functional understanding of the structure of tRNA, but will also shed light on the molecular contacts between EF-Tu and tRNA. This insight will form the basis upon which modeling of the crystal structures of EF-Tu and tRNA can be established to understand the mechanism of EF-Tu and tRNA interaction that underlies the fidelity of genetic information transfer.