Stopped-flow Kinetic Analysis of eIF4E and Phosphorylated eIF4E Binding to Cap Analogs and Capped Oligoribonucleotides

Recruitment of eukaryotic mRNA to the 48 S initiation complex is rate-limiting for protein synthesis under normal conditions. Binding of the 5′ -terminal cap structure of mRNA to eIF4E is a critical event during this process. Mammalian eIF4E is phosphorylated at Ser-209 by Mnk1 and Mnk2 kinases. We investigated the interaction of both eIF4E and phosphorylated eIF4E (eIF4E(P)) with cap analogs and capped oligoribonucleotides by stopped-flow kinetics. For m7GpppG, the rate constant of association, kon, was dependent on ionic strength, decreasing progressively up to 350 mm KCl, but the rate constant of dissociation, koff, was independent of ionic strength. Phosphorylation of eIF4E decreased kon by 2.1-2.3-fold at 50-100 mm KCl but had progressively less effect at higher ionic strengths, being negligible at 350 mm. Contrary to published evidence, eIF4E phosphorylation had no effect on koff. Several observations supported a simple one-step binding mechanism, in contrast to published reports of a two-step mechanism. The kinetic function that best fit the data changed from single- to double-exponential as the eIF4E concentration was increased. However, measuring koff for dissociation of a pre-formed eIF4E·m7GpppG complex suggested that the double-exponential kinetics were caused by dissociation of eIF4E dimers, not a two-step mechanism. Addition of a 12-nucleotide chain to the cap structure increased affinity at high ionic strength for both eIF4E (24-fold) and eIF4E(P) (7-fold), primarily due to a decrease in koff. This suggests that additional stabilizing interactions between capped oligoribonucleotides and eIF4E, which do not occur with cap analogs alone, act to slow dissociation.

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