10] Identifying primary translation products: Use of N-formylmethionyl-tRNA and prevention of NH2-terminal acetylation

Abstract

The synthesis of all proteins is initiated with methionine, but the NH 2 terminus of only a small percentage of mature proteins is methionine. Because of polypeptide cleavage and NH 2 -terminal modification, special techniques are required to identify and sequence primary translation products. This chapter describes the techniques, preparation of N-Formyl[ 35 S]Met-tRNA Met f , preparation of [ 35 S]Met-tRNA Met m and an alternative procedure for preparation of [ 35 S]Met-tRNA Met f , and prevention of NH 2 -terminal acetylation. After isolation of the protein, the formyl group can be removed by mild acid hydrolysis for sequencing. If only labeled Met-tRNA Met is desired, the [ 35 S]Met-tRNA Met f can be specifically deacylated by using the E. coli extract. The major problem is competition between endogenous, unlabeled Met-tRNA Met f and added N-formyl[ 35 S]Met-tRNA Met f ; thus, one should add as much labeled tRNA as possible without inhibiting translation significantly. As the N-terminal acetyl group is derived from acetyl-CoA, one strategy for prevention of acetylation is to reduce the pool of acetyl-CoA using a metabolic trap, which takes advantage of the fact that acetyl-CoA can be effectively depleted by addition of oxaloacetate and citrate synthase to the lysate.