Studies on the role of eukaryotic nucleotide exchange factor in polypeptide chain initiation.
Interactions of eukaryotic 5-dimethylaminonaphthalene-1-sulfonyl-initiation factor 2 (eIF-2) from rabbit reticulocytes and the guanine nucleotide exchange factor ( GEF ), Met-tRNAf, GTP, and GDP were monitored by changes in fluorescence anisotropy and radioactive filtration assays. At 1 mM Mg2+, radioactive filtration assays demonstrate that GEF is necessary for nucleotide exchange. We did not observe a GDP dependence in the association reaction of eIF-2 X GEF for GDP concentrations from 0.01 to 20 microM. This is in disagreement with the model: eIF-2 X GDP + GEF in equilibrium eIF-2 X GEF + GDP. The addition of GTP caused a decrease in fluorescence anisotropy which is interpreted as a dissociation of eIF-2 X GEF . We propose an asymmetrical model of ternary complex (eIF-2 X GTP X Met-tRNAf) formation where 1) GDP does not displace GEF and 2) GTP replaces GEF and presumably GDP. For reticulocyte eIF-2, phosphorylation of the alpha subunit greatly inhibits protein synthesis. This inhibition derives neither from failure of GEF to bind to eIF-2(alpha P) nor from greatly enhanced binding of GEF . The inhibition results from the requirement of very high levels of GTP (100 microM) to dissociate the eIF-2(alpha P) X GEF complex.
D015852:Eukaryotic Initiation Factor-2; D005454:Fluorescence Polarization; D019204:GTP-Binding Proteins; D006153:Guanosine Diphosphate / Q000378:metabolism; D007700:Kinetics; D008954:Models, Biological; D010442:Peptide Chain Initiation, Translational; D010448:Peptide Initiation Factors / Q000378:metabolism*; D010766:Phosphorylation; D011506:Proteins / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000378:metabolism*
D015852:Eukaryotic Initiation Factor-2; D010448:Peptide Initiation Factors; D011506:Proteins; D011956:Receptors, Cell Surface; D006153:Guanosine Diphosphate; D019204:GTP-Binding Proteins