Heimark R L, Hershey J W, Traut R R
J Biol Chem. 1976 Dec 25;251(24):7779-84.
Initiation factor IF2 bound to the 70 S initiation complex with 5'-guanylyl methylenediphosphonate was treated with the cross-linking reagent, dimethyl-3,3'-dithiobispropionimidate. Covalent cross-linking of the factor to ribosomes was demonstrated by stabilization of initiation factor IF2-70S complexes during centrifugation at high salt concentrations. Specific cross-linking of the factor to the 50 S proteins L7/L12 was shown by: (a) co-precipitation of the 50 S proteins L7/L12 by antibodies made against initiation factor IF2 and (b) the appearance of radioactive bands containing [32P]phosphoryl initiation factor IF2 in regions of elevated molecular weight following polyacrylamide/dodecyl sulfate gel electrophoresis. One major band had an apparent molecular weight of 132,000, consistent with its being a dimer containing one copy of L7/L12 (13,000) and initiation factor IF2 (120,000). This cross-linked species was shown to contain [32P]phosphoryl initiation factor IF2 and 35S-labeled L7/L12 in separate experiments. It is concluded that L7/L12, which were shown previously to be required for the binding of the factor, are located at or near the initiation factor IF2 binding site on the 70 S initiation complex.
