The long term goal of this project is to determine the nature of site specific structural and functional interactions of transfer RNA (tRNA) with other nucleic acids and proteins particularly with regard to the structure found around the modified nucleosides. Site specific heteronuclear enrichment of modified nucleosides combined with proton and heteronuclear NMR techniques (for the probing of local conformations and structure not readily accessible by other methods) will be carried out. A unique E. coli plasmid has been constructed into which any chemically synthesized tRNA gene can be placed for overproduction of the tRNA in vivo. Both 13C-enriched wild type and designed mutant tRNAs with site selected nucleoside changes can be produced in quantity for analysis of biochemical function and for NMR studies. The interaction of tRNA with proteins and nucleic acids depends upon the tRNA molecule's conformation in solution and its specific flexibilities, neither of which are fully understood. A study of tRNA conformation and site specific mobility is also important for a comparison of E. coli and yeast tRNA structures. The advantage of NMR over other physical techniques in studying macromolecules in solution is that signals are obtainable from reporter groups in many different parts of the molecules simultaneously.
