Direct visualization of unwinding activity of Duplex RNA by Dhpa, a dead box helicase, at single molecule resolution

H. Arnon, M. Ohad, Barak Akabayov, Serdoc Irit

Research output: Book/ReportBookpeer-review

Abstract

The Eschericia coli protein, DbpA, is unique in its subclass of DEAD box RNA helicases because it possesses ATPase specific activity toward the peptidyl transferase center in 23S rRNA. Although its remarkable ATPase activity is well defined toward various substrates, its RNA helicase activity remained to be characterized. We show by using biochemical assays and atomic force microscopy (AFM) that DbpA exhibits ATP-stimulated unwinding activity of RNA duplex regardless of its primary sequence. The work presents an attempt to investigate the action of DEAD-box proteins by a single-molecule visualization methodology. Our AFM images enabled us to directly observe the unwinding reaction of a DEAD box helicase on long stretches of double stranded RNA (dsRNA). Specifically, we could differentiate between defined steps in the unwinding reaction. Recent studies have questioned the designation of DbpA, in particular, and DEAD box proteins in general as RNA helicases. However, accumulated evidences and the results reported here suggest that these proteins are indeed helicases that resemble in many aspects the DNA helicases. Although the biological function of DbpA and many RNA helicases from the DEAD box family remain to be elucidated, this work provides means to understand the structural bases of the motoric and enzymatic activity of DEAD box proteins. In addition, application of AFM to the study of these motor proteins open a new way to investigate the intimate relationship between these enzymes and their co-factors needed for the biological catalysis.
Original languageEnglish
DOIs
StatePublished - 1 Feb 2002

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