Single-Molecule Studies of the Effects of Small Compounds on the Activity of Translation Initiation Factor eIF4A

Abstract

The translation initiation factor eIF4A is the prototypical DEAD-box RNA helicase as a subunit of eIF4F. It facilitates the binding and scanning of the ribosome via unwinding secondary structures in the 5’ UTR of mRNAs during translation initiation. Here single-molecule Fluorescence Resonance Energy Transfer (sm-FRET) assay for both structured and unstructured substrates is performed and it shows that eIF4A unwinds the substrates in a discrete step manner although it is a highly nonprocessive motor. It also shows that eIF4A is a bidirectional helicase and the step size is about 6 base pair. Pateamine A and silvestrol are small-molecule modulators of eIF4A activity and they are identified as potent inhibitors of translation. It was demonstrated that Pateamine A and silvestrol act as a chemical inducer of dimerization and promote the interaction between eIF4A and RNA, however the molecular mechanisms by which they regulate eIF4A activities still remain elusive. Our sm-FRET assay shows that both pateamine A and silvestrol stimulate the helicase activity of eIF4A. Understanding how the processivity of eIF4A is influenced by these molecules will help regulating the translation at the initiation step.