Abstract: DEAD-box proteins are believed to participate in the folding of RNA by destabilizing RNA secondary or tertiary structures. Although these proteins bind and hydrolyze ATP, the mechanism by which nucleotide hydrolysis is coupled to helix destabilization may vary among different DEAD-box proteins. To investigate their abilities to disrupt helices and couple ATP hydrolysis to unwinding, we assayed the Saccharomyces cerevisiae ribosomal DEAD-box proteins, Dbp3p, Dbp4p, Rok1p, and Rrp3p utilizing a series of RNA substrates containing a short duplex and either a 5' or 3' single-stranded region. All four proteins unwound a 10 bp helix in vitro in the presence of ATP; however, significant dissociation of longer helices was not observed. While Dbp3p did not require a single-stranded extension to disrupt a helix, the unwinding activities of Dbp4p, Rok1p, and Rrp3p were substantially stimulated by either a 5' or 3' single-stranded extension. Interestingly, these proteins showed a clear length dependency with 3' extensions that was not observed with 5' extensions, suggesting that they bind substrates with a preferred orientation. In the presence of AMPPNP or ADP, all four proteins displayed displacement activity suggesting that nucleotide binding is sufficient to facilitate duplex disruption. Further enhancement of the strand displacement rate in the presence of ATP was observed for only Dbp3p and Rrp3p.