The RNA helicase Mtr4p is a duplex-sensing translocase

The conserved Saccharomyces cerevisiae Ski2-like RNA helicase Mtr4p plays essential roles in eukaryotic nuclear RNA processing. RNA helicase activity of Mtr4p is critical for biological functions of the enzyme, but the molecular basis for RNA unwinding is not understood. Here, single-molecule high-resolution optical trapping measurements reveal that Mtr4p unwinds RNA duplexes by 3'-to-5' translocation on the loading strand, that strand separation occurs in discrete steps of 6 base pairs and that a single Mtr4p molecule performs consecutive unwinding steps. We further show that RNA unwinding by Mtr4p requires interaction with upstream RNA duplex. Inclusion of Mtr4p within the TRAMP complex increases the rate constant for unwinding initiation but does not change the characteristics of Mtr4p's helicase mechanism. Our data indicate that Mtr4p utilizes a previously unknown unwinding mode that combines aspects of canonical translocating helicases and non-canonical duplex-sensing helicases, thereby restricting directional translocation to duplex regions.