eIF4A is the archetypal member of the DEAD box family of proteins and has been proposed to use the energy from ATP hydrolysis to unwind structures in the 5'-untranslated regions of eukaryotic mRNAs during translation initiation. As a step toward understanding the mechanism of action of this class of enzymes, a minimal kinetic and thermodynamic framework for the RNA- activated ATPase function has been established for eIF4A. The enzyme's affinity for ssRNA is modulated by the binding of ATP·Mg2+ and ADP·Mg2+: the affinity of the E·ATP complex for ssRNA is approximately 40-fold higher than that of the E·ADP complex. The enzyme binds its substrates in a random manner: contrary to previous suggestions, neither ATP binding nor hydrolysis is required for binding of single-stranded RNA. The presence or absence of the χ-phosphate on the bound nucleotide acts as a switch that modulates the enzyme's structure and ssRNA affinity. The data presented in this and the following paper in this issue suggest that ATP binding and hydrolysis produce a cycle of conformational and RNA affinity changes in eIF4A. Such cycles may be used by DEAD box proteins to transduce the energy from ATP hydrolysis into physical work, thereby allowing each member of this family to rearrange its RNA or RNA·protein target.
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