| Abstract: | A method for unambiguously determining the initiator methionine residue and the adjacent NH2-terminal amino acid sequence of cell-free translation products of eukaryotic messenger RNA is described. In this procedure, the NH2 termini of nascent peptides are blocked by incorporating labeled formylmethionine instead of methionine, using yeast initiator tRNA in the wheat germ cell-free system. After immunoprecipitation of the desired product the radiolabeled material is treated with dansyl-Cl to irreversibly block all remaining free amino groups. The material is then deformylated by mild acid hydrolysis and subjected to automated Edman degradation. Only those products that had been synthesized with formylmethionine residues at their NH2-termini can then give rise to labeled phenylthiohydantoin derivatives during degradation. Using this method, we have defined the initiation sites in both rat preproinsulin and pregrowth hormone messenger RNAs. |
