Visualization of unwinding activity of duplex RNA by DbpA, a DEAD box helicase, at single-molecule resolution by atomic force microscopy

The Escherichia coli protein DbpA is unique in its subclass of DEAD box RNA helicases, because it possesses ATPase-specific activity toward the peptid yl transferase center in 23S rRNA, Although its remarkable ATPase activity had been well defined toward various substrates, its RNA helicase activity remained to be characterized. Herein, we show by using biochemical assays a nd atomic force microscopy that DbpA exhibits ATP-stimulated unwinding acti vity of RNA duplex regardless of its primary sequence. This work presents a n attempt to investigate the action of DEAD box proteins by a single-molecu le visualization methodology. Our atomic force microscopy images enabled us to observe directly the unwinding reaction of a DEAD box helicase on long stretches of double-stranded RNA. Specifically, we could differentiate betw een the binding of DbpA to RNA in the absence of ATP and the formation of a Y-shaped intermediate after its progression through double-stranded RNA in the presence of ATP, Recent studies have questioned the designation of Dbp A, in particular, and DEAD box proteins in general as RNA helicases, Howeve r, accumulated evidence and the results reported herein suggest that these proteins are indeed helicases that resemble in many aspects the DNA helicas es.