The Site of Ef 2 Interaction of the Eukaryotic Ribosome

Irvin, James

Abstract

The interaction of elongation factor 2(EF 2) with the eukaryotic ribosome has been studied for a number of years but very little is known about the ribosomal components with which it interacts nor how it catalyzes the translocation step of the elongation cycle. It is proposed to investigate the ribosomal interaction of EF 2' by preparing cross-linked complexes of the factor with the ribosome and identify the ribosomal components which interact with EF 2. These studies will use EF 2 prederivatized with a heterobifunctional cross-linking reagent attached at a number of different sites on the factor which will be activated for cross-linking to the ribosome by photolysis after isolation of preformed EF 2-ribosome complexes formed in the presence of guanine nucleotides. This methodology should reduce the amount of nonspecific cross-linking and allow for the determination of the relative position of the ribosomal components cross-linked to EF 2 under conditions which should discrimate its pre- and post-translocation site of binding. The same methodology will be used to examine the interaction of ADP-ribosylated EF 2, formed by the catalytic action of diphtheria toxin, with ribosomes and the interaction of ribosomes damaged by ribosomal inactivating proteins which catalytically inactivate eukaryotic ribosomes by an unknown mechanism. Since the process of protein synthesis is essential for all growing cells it is of primary importance that the process be understood in detail for continued elucidation of the life processes. Also the determination of the mechanism by which diphtheria toxin and the ribosomal inactivating proteins inhibit protein synthesis is of prime importance since these agents are being used to construct immunotoxins directed against cancer cells for possible chemotherapy and such agents are being tested as immunosuppressive agents to aid organ transplantation. The more that is known concerning their action should aid in their therapeutic applications and provide additional information about the function of the eukaryotic ribosome.