Poster abstracts

Poster number 113 submitted by Eric Patrick

The eukaryotic RNA helicase Mtr4p is a duplex-sensing translocase

Eric M. Patrick (Department of Physics and Astronomy, Michigan State University, East Lansing MI, U.S.A), Sukanya Srinivasan (Center for RNA Molecular Biology and Department of Biochemistry, Case Western University, Cleveland OH, U.S.A), Eckhard Jankowsky (Center for RNA Molecular Biology and Department of Biochemistry, Case Western University, Cleveland OH, U.S.A), Matthew J. Comstock (Department of Physics and Astronomy, Michigan State University, East Lansing MI, U.S.A)

Abstract:
The conserved 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 by Mtr4p 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 protomer 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, to restrict directional translocation to duplex regions. We have additionally compared Mtr4p unwinding to the non-translocating DEAD box RNA helicase, Ded1p, which shows limited RNA unwinding followed by RNA rezipping. Finally, we present recent high-resolution fleezers measurements of TRAMP unwinding and polymerase activity observed simultaneously. Polyadenylation of the RNA substrate free 3′ overhang by TRAMP is observed via the hybridization of fluorescently labeled oligonucleotide probes.

Keywords: RNA helicase, Ski2-like, Translocase