Initiation of the Polypeptide Chain by Reticulocyte Cell‐Free Systems

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Abstract
In order to elucidate the mechanism of action of inhibitors that block the initiation of protein synthesis in mammalian systems, we have studied the following steps: (a) formation of the ternary complex Met-tRNAr-IF-E2-GTP, (b) binding of the initiator Met-tRNAf to the 40-S ribosomal subunit in the presence of initiation factors and dependent or not on the addition of mRNA, (c) formation of the initiation complex with 80-S ribosomes and (d) formation of the first peptide bond. Adrenochrome, aurintricarboxylic acid, polydextran sulphate, pyrochatechol violet and showdomycin block the formation of the ternary complex Met-tRNAf-IF-E2-GTP. Edeine A1, aurintricarboxylic acid and polydextran sulphate block the binding of the mRNA to the 40-S ribosomal subunit. Pactamycin induces the formation of stable smaller initiation complexes which are unable to go through the subsequent steps of initiation. Stimulation of the binding of the initiator Met-tRNAf to the 80-S ribosome in the presence of initiation factors is observed with sparsomycin and antibiotics of the sesquiterpene family (verrucarin A, trichodermin and trichothecin). However, these antibiotics block the reaction of the bound Met-tRNAf with puromycin. Narciclasine has no effect on the binding of the initiator to the ribosome but strongly blocks its reaction with puromycin. We have developed a simple technique to detect the Met-tRNAf-40-S-subunit-poly(A, G, U) initiation complexes by chromatography on Sepharose 6B columns. The requirements for the formation of such complexes measured by this technique and its comparison with the sucrose gradient centrifugation method are described.