Boston University

RNA helicase activity probed at the single molecule level

RNA helicases are molecular motors that are involved in most aspects of RNA metabolism. Eukoryotic Initiation Factor eIF4A is the prototype protein of the DEAD box family of RNA helicases. It is thought to unwinds structures in the 5′-untranslated regions of eukaryotic mRNAs in an ATP dependent manner, and in conjunction with other translation factors it prepares mRNA templates for cap-dependent ribosome recruitment during translation initiation.

We employ single molecule techniques in order to delineate eIF4A (as well as the complex eIF4F) unwinding activity. The proteins are expressed and purified with single point mutations, to enable binding of fluorescent tags. A combination of single molecule fluorescence resonance energy transfer (FRET) and nanopore experiments is used to address binding conformations and activity of the helicases. Our FRET measurements explore the location of eIF4A and its sliding motion along the RNA. The kinetics of the unwinding mechanism and its dependence on RNA sequence structure is also investigated. The results will enable better characterization of eIF4A helicase activity and protein-RNA interactions. This work is performed in collaboration with Jerry Pelletier's group ( and Nahum Sonenberg's group ( at McGill university.


HFSPNational Institute of Health
Figure 1