RNA aptamers to initiation factor 4A helicase hinder cap-dependent translation by blocking ATP hydrolysis.
The mammalian translation initiation factor 4A (eIF4A) is a prototype member of the DEAD-box RNA helicase family that couples ATPase activity to RNA binding and unwinding. In the crystal form, eIF4A has a distended "dumbbell" structure consisting of two domains, which probably undergo a conformational change, on binding ATP, to form a compact, functional structure via the juxtaposition of the two domains. Moreover, additional conformational changes between two domains may be involved in the ATPase and helicase activity of eIF4A. The molecular basis of these conformational changes, however, is not understood. Here, we generated RNA aptamers with high affinity for eIF4A by in vitro RNA selection-amplification. On binding, the RNAs inhibit ATP hydrolysis. One class of RNAs contains members that exhibit dissociation constant of 27 nM for eIF4A and severely inhibit cap-dependent in vitro translation. The binding affinity was increased on Arg substitution in the conserved motif Ia of eIF4A, which probably improves a predicted arginine network to bind RNA substrates. Selected RNAs, however, failed to bind either domain of eIF4A that had been split at the linker site. These findings suggest that the selected RNAs interact cooperatively with both domains of eIF4A, either in the dumbbell or the compact form, and entrap it into a dead-end conformation, probably by blocking the conformational change of eIF4A. The selected RNAs, therefore, represent a new class of specific inhibitors that are suitable for the analysis of eukaryotic initiation, and which pose a potential therapeutic against malignancies that are caused by aberrant translational control.
Oguro
Akihiro
A
Department of Basic Medical Sciences, Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo 108-8639, Japan.
Ohtsu
Takashi
T
Svitkin
Yuri V
YV
Sonenberg
Nahum
N
Nakamura
Yoshikazu
Y
D000255:Adenosine Triphosphate / Q000378:metabolism; D001483:Base Sequence; D039601:Eukaryotic Initiation Factor-4A / Q000378:metabolism*; D039603:Eukaryotic Initiation Factor-4G / Q000378:metabolism; D008969:Molecular Sequence Data; D014176:Protein Biosynthesis / Q000502:physiology*; D012313:RNA / Q000378:metabolism*; D017423:Sequence Analysis, RNA
D039603:Eukaryotic Initiation Factor-4G; D012313:RNA; D000255:Adenosine Triphosphate; D039601:Eukaryotic Initiation Factor-4A