Biochemical and kinetic characterization of the RNA helicase activity of eukaryotic initiation factor 4A

Eukaryotic initiation factor (eIF) 4A is the prototypic member of the DEAD box family of proteins and has been proposed to act as an RNA helicase to u nwind secondary structure in the 5'-untranslated region of eukaryotic mRNAs , Previous studies have shown that the RNA helicase activity of eIF4A is de pendent on the presence of a second initiation factor, eIF4B, In this repor t, eIF4A has been demonstrated to function independently of eIF4B as an ATP -dependent RNA helicase, The bio chemical and kinetic properties of this ac tivity were examined. By using a family of RNA duplexes with an unstructure d single-stranded region followed by a duplex region of increasing length a nd stability, it was observed that the initial rate of duplex unwinding dec reased with increasing stability of the duplex. Furthermore, the maximum am ount of duplex unwound also decreased with increasing stability. Results su ggest that eIF4A acts in a non-processive manner. eIF4B and eIF4H were show n to stimulate the helicase activity of eIF4A, allowing eIF4A to unwind lon ger, more stable duplexes with both an increase in initial rate and maximum amount of duplex unwound. A simple kinetic model is proposed to explain th e mechanism by which eIF4A unwinds RNA duplex structures in an ATP-dependen t manner.