Poly(A)‐Binding Protein Affects the Kinetics of Tobacco Etch Virus Pseudoknot RNA Binding to Wheat germ Translation Initiation Factor eIF4F

Previous studies indicated that the pseudoknot (PK) of tobacco etch virus (TEV) RNA is necessary to promote cap‐independent translation (Zeenko and Gallie, 2005, J.B.C; 280, 26813‐26824). We investigated the effect of poly(A)‐binding protein (PABP) on the PK1 RNA binding to eIF4F in the absence and presence of eIF4B. Equilibrium studies of PK1 RNA binding to eIF4F showed ~ 2‐fold stronger affinity in the presence of PABP. Addition of PABP and eIF4B to the eIF4F enhances binding affinity ~3.5‐fold as compared to eIF4F binding to PK1 RNA. Binding of eIF4F·PABP and eIF4F·4B·PABP to PK1 RNA is both enthalpy and entropy‐favorable. Further, kinetic analysis of eIF4F, eIF4F·PABP and eIF4F·4B·PABP with PK1 RNA and the temperature dependence of this reaction were measured and compared. The stopped‐flow fluorescence anisotropy data showed that the observed rate constant for the binding of PK1 RNA increased linearly with an increase in eIF4F, eIF4F·PABP or eIF4F·4B·PABP initiation factor concentration. The association rate constant (k on ) for PK1 binding was ~ 2.8‐fold faster for eIF4F·4B·PABP than eIF4F. The dissociation rate constant (k off ) was ~1.5‐fold slower for eIF4F‐PK1 in the presence of eIF4B and PABP. The activation energy of the PK1 RNA binding reaction was markedly reduced in the presence of PABP and eIF4B. The enhancement of k on , lowering of k off , and reduction in energy barrier for the formation of eIF4F·4B·PABP‐PK1 complex suggests a more stable platform for efficient translation of protein synthesis. These results demonstrate the first direct kinetic measurements of plant translation initiation factors binding to an internal ribosome entry site (IRES).