Abstract
Previous kinetic binding studies of wheat germ protein synthesis eukaryotic initiation factor iso4F (eIFiso4F) and its subunit, eIF4E, with m(7)GTP and mRNA analogues indicated that binding occurred by a two-step process with the first step being too fast to measure by stopped-flow techniques (). Further equilibrium studies showed that poly(A)-binding protein (PABP) enhanced the cap binding of eIFiso4F about 40-fold. The kinetic effects of PABP on cap binding and the temperature dependence of this reaction were measured and compared. Fluorescence stopped-flow studies of the PABP.eIFiso4F protein complex with cap show a concentration-independent conformational change. PABP did not significantly increase the rate of the conformational change, and because the initial second-order binding is essentially diffusion-controlled, the enhancement of cap affinity must reside in the dissociation rate. The dissociation rate was more than 5-fold slower in the presence of PABP. The temperature dependence of the cap binding reaction was markedly reduced in the presence of PABP. The reduced energy barrier for formation of a cap.eIFiso4F complex suggests a more stable platform for further initiation complex formation and a possible means of adapting to varying temperature conditions.
Highlights
Previous kinetic binding studies of wheat germ protein synthesis eukaryotic initiation factor iso4F and its subunit, eIF4E, with m7GTP and mRNA analogues indicated that binding occurred by a two-step process with the first step being too fast to measure by stopped-flow techniques [1]
Time course data were fitted by nonlinear regression analysis assuming a single exponential change described by ⌬Ft ϭ ⌬Fρ (1 Ϫ exp(Ϫkobst)) [16], where ⌬Ft is that fluorescence observed at any time, t, and the final fluorescence when the reaction achieves equilibrium, ⌬Fρ, is the difference between Ant-m7GTP fluorescence at zero time and at equilibrium, and kobs is the observed first-order rate constant
Under the pseudo first-order conditions, where Ant-m7GTP was in excess, the observed rate constant is predicted to be a linear function of the concentration of Ant-m7GTP
Summary
WHEAT GERM POLY(A)-BINDING PROTEIN LOWERS THE ACTIVATION ENERGY BARRIER TO INITIATION COMPLEX FORMATION*. The increase in binding affinity of the PABP1⁄7eIF4F complex for both cap analogs and poly(A) leads us to further investigate the kinetics and mechanism of these interactions. We report the effects of PABP on the binding kinetics of cap analog binding to eIFiso4F and the temperature dependence of these reactions. These data indicate that a large part of the enhancement in binding affinity resides in the dissociation rate and that one of the significant effects of PABP is to lower the Arrehenius activation energy of the eIF4F complex
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