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 unwind secondary structure in the 5'-untranslated region of eukaryotic mRNAs. Previous studies have shown that the RNA helicase activity of eIF4A is dependent on the presence of a second initiation factor, eIF4B. In this report, eIF4A has been demonstrated to function independently of eIF4B as an ATP-dependent RNA helicase. The biochemical and kinetic properties of this activity were examined. By using a family of RNA duplexes with an unstructured single-stranded region followed by a duplex region of increasing length and stability, it was observed that the initial rate of duplex unwinding decreased with increasing stability of the duplex. Furthermore, the maximum amount of duplex unwound also decreased with increasing stability. Results suggest that eIF4A acts in a non-processive manner. eIF4B and eIF4H were shown to stimulate the helicase activity of eIF4A, allowing eIF4A to unwind longer, more stable duplexes with both an increase in initial rate and maximum amount of duplex unwound. A simple kinetic model is proposed to explain the mechanism by which eIF4A unwinds RNA duplex structures in an ATP-dependent manner.
Rogers
G W
GW
Department of Biochemistry, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106-4935, USA.
Richter
N J
NJ
Merrick
W C
WC
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D017931:DNA Primers; D039601:Eukaryotic Initiation Factor-4A; D007700:Kinetics; D010448:Peptide Initiation Factors / Q000378:metabolism*; D020365:RNA Helicases / Q000378:metabolism*; D011817:Rabbits; D013379:Substrate Specificity
D017931:DNA Primers; D010448:Peptide Initiation Factors; D000255:Adenosine Triphosphate; D039601:Eukaryotic Initiation Factor-4A; D020365:RNA Helicases