THE DEAD BOX PROTEIN EIF4A - 1 - A MINIMAL KINETIC AND THERMODYNAMIC FRAMEWORK REVEALS COUPLED BINDING OF RNA AND NUCLEOTIDE

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, str uctures in the 5'-untranslated regions of eukaryotic mRNAs during tran slation initiation. As a step toward understanding the mechanism of ac tion of this class of enzymes, a minimal kinetic and thermodynamic fra mework for;he RNA-activated ATPase function has been established for e IF4A. The enzyme's affinity for ssRNA is modulated by the binding of A TP.Mg2+ and ADP.Mg2+: the affinity of the E.ATP complex for ssRNA is a pproximately 40-fold higher than that of the E.ADP complex, The enzyme binds its substrates in a random manner; contrary to previous suggest ions, neither ATP binding nor hydrolysis is required for binding of si ngle-stranded RNA. The bo presence or absence of the gamma-phosphate o n the bound nucleotide acts as a switch that modulates the enzyme's st ructure and ssRNA affinity. The data presented in this and the followi ng paper in this issue suggest that ATP binding and hydrolysis produce a cycle of conformational and RNA affinity changes in eIF4A, Such cyc les may be used by DEAD box proteins to transduce the energy from ATP hydrolysis into physical work, thereby allowing each member of this fa mily to rearrange its RNA or RNA protein target.