This proposal is to investigate that Tetrahymena ribozyme catalyze peptide bond formation reactions. We hypothesize that in Tetrahymena ribozyme, the catalytic core for reactions of phosphate diesters might possess a catalytic capability for reactions at carbon centers. We propose to use the oligonucleotide of 3'-aminoacyl-CCCUCU linking with a variety of blocked amino acids as a substrate to test this idea. We will probe peptidyl transfer reactions such as the transfer of an amino acid to arginine to form a dipeptide, to 3'-deoxy-3'-amimo-guanosine-5'- monophosphate, or to 3'-N-aminoacyl-3'-deoxy-3'-amino-guanosine to form a peptidyl bond, or to guanosine. We will examine the specificity of the ribozyme for the amino acid moiety of CCCUCU-aa, and determine whether the amino acid affects the equilibrium binding constant or the rate of the chemical step. We will also test the effects of various divalent metal ions on ribozyme catalysis and employ mechanistic studies including measurement of the rate of the chemical step or a function of pH to elucidate the mechanism of the peptidyl transfer by RNA. Furthermore, in vitro selection and in vitro evolution methods represent powerful tools for isolating functional RNA molecules. We also can apply in vitro selection techniques to obtain the novel catalytic RNAs for peptidyl transfer reactions and other chemical reactions.
