Protein synthesis in rabbit reticulocytes: Mg2+-inhibition of ternary complex (Met-tRNA(f).eIF-2.GTP) formation by reticulocyte eIF-2.
There are conflicting reports regarding Mg2+-inhibition of ternary complex formation by reticulocyte eIF-2. Several laboratories have reported that eIF-2 is isolated as eIF-2.GDP and Mg2+ inhibits ternary complex formation, as in the presence of Mg2+, 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 Mg2+ has no effect on eIF-2 activity and eIF-2 forms near stoichiometric amount of ternary complex in the presence of Mg2+. In this paper, we provide evidence which suggests that the Mg2+-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 Mg2+ in the reaction mixture and there was no free Mg2+ 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 Mg2+ inhibition.
D000818:Animals; D015852:Eukaryotic Initiation Factor-2; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism*; D046911:Macromolecular Substances; D008274:Magnesium / Q000494:pharmacology*; D010442:Peptide Chain Initiation, Translational; D010448:Peptide Initiation Factors / Q000378:metabolism*; D014176:Protein Biosynthesis; D011506:Proteins / Q000378:metabolism*; D012346:RNA, Transfer, Amino Acyl / Q000378:metabolism*; D012358:RNA, Transfer, Met; D011817:Rabbits; D012156:Reticulocytes / Q000378:metabolism*
D015852:Eukaryotic Initiation Factor-2; D046911:Macromolecular Substances; D010448:Peptide Initiation Factors; D011506:Proteins; D012346:RNA, Transfer, Amino Acyl; D012358:RNA, Transfer, Met; C024988:tRNA, formylmethionine-; D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate; D008274:Magnesium