CHARACTERIZATION OF THE ATP-DEPENDENT BINDING OF WHEAT-GERM PROTEIN-SYNTHESIS INITIATION FACTOR-EIF-(ISO)4F AND FACTOR-EIF-4A TO MESSENGER-RNA

The ATP-dependent binding of wheat germ protein synthesis initiation f actors eIF-(iso)4F and eIF-4A to an oligoribonucleotide has been inves tigated by direct fluorescence titration techniques. In addition, the effect of ATP on the interaction between another cap-binding initiatio n factor, eIF-4F, and eIF-4A was studied using the same methods. Compa rison of the equilibrium association constants (K(eq)) indicate that 1 ) hydrolyzable ATP affects the affinity of eIF-(iso)4F for eIF-4A, reg ardless of whether or not mRNA was previously bound to the eIF-(iso)4F ; in contrast, ATP had no effect on the eIF-(iso)4F/oligoribonucleotid e interaction; 2) in the presence of ATP, the binding of the binary eI F-(iso)4F.eIF-4A complex to the oligoribonucleotide is of similar affi nity as the binding of the oligoribonucleotide to the eIF-(iso)4F alon e; the stoichiometry of this ternary eIF-(iso)4F.eIF-4A-mRNA complex w as found to be 1:1:1; and 3) a similar ATP effect is observed for the eIF-4F/eIF-4A interaction as for the eIF-(iso)4F.eIF-4A complex.