Protein synthesis in rabbit reticulocytes: Mg 2+ - inhibition of ternary complex (met-tRNA f·eIF-2·GTP) formation by reticulocyte eIF-2

Abstract

There are conflicting reports regarding Mg 2+-inhibition of ternary complex formation by reticulocyte eIF-2. Several laboratories have reported that eIF-2 is isolated as eIF-2·GDP and Mg 2+ inhibits ternary complex formation, as in the presence of Mg 2+, GDP remains tightly bound to eIF-2 and prevents ternary complex formation. A protein factor, GEF is necessary for GDP displacement and subsequent ternary complex formation. Other laboratories have reported that Mg 2+ has no effect on eIF-2 activity and eIF-2 forms near stoichiometric amount of ternary complex in the presence of Mg 2+. In this paper, we provide evidence which suggests that the Mg 2+-insensitive eIF-2 activity as reported by several laboratories might have been the result of the use of high Met-tRNA f concentrations in their assays as the nucleotides in excess tRNA bound Mg 2+ in the reaction mixture and there was no free Mg 2+ available to inhibit eIF-2 activity. Our data will show that the addition of excess tRNA promotes non-enzymatic GDP displacement from eIF-2·GDP and relieves Mg 2+ inhibition.