Investigations of Reactions of Physiological Importance

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DESCRIPTION (provided by applicant): The genetic code was established over two billion years ago. Transfer RNAs and aminoacyl tRNA synthetases are at the heart of the code. The synthetases catalyze the attachment of each of the twenty amino acids to their cognate tRNA. Because each tRNA harbors the anticodon triplet for a particular amino acid, the aminoacylation reaction establishes the connection between a nucleotide triplet and a particular amino acid. Transfer RNAs are composed of two major domains that are conserved through evolution. The domains are arranged in three dimensions in an L-shaped structure. One domain is the minihelix, a 12 bp hairpin helix that at the 3'-end is single-stranded and contains the amino acid attachment site. The second domain is positioned at right angles to the first and is a stem-biloop, with one of the loops encoding the anticodon triplet. These two domains are thought by many to have arisen independently. Experiments are designed to explore, in effect, how to make the transition from a minihelix to the genetic code, using noncovalent interactions between the two domains as a way to connect the triplets of the code to an attached amino acid. Because aminoacylated RNA minihelices are considered possible progenitors of aminoacyl tRNAs, a second goal is to establish systems for peptide synthesis based on minihelices. These systems are meant to illustrate at least a few ways to make the transition from the putative RNA world to the theatre of proteins. They also recreate some features of contemporary protein synthesis, including the possibility of reactions dependent on an RNA template. A third objective is to understand the RNA-dependent amino acid discrimination that makes possible the modern universal code. Specifically, experiments are designed to elucidate how subtle information in tRNAs is used with a second active site in synthetases to perfect the code. Because tRNA synthetases as essential proteins are targets for new anti-infectives, and because fragments of specific synthetases in mammalian cells are involved in signal transduction pathways and therefore can be exploited for therapeutic applications, the proposed research strengthens the base of knowledge upon which health applications can be made.