Poster abstracts
Poster number 128 submitted by Kasun Senanayake
Step-By-Step Binding and Unwinding Of RNA By A Ded1p Triplet Revealed By Combined High-Resolution Tweezers And Fluorescence Measurements
Kasun B. Senanayake (Department of Physics and Astronomy, Michigan State University, East Lansing MI, U.S.A.), Rajeev Yadav (Department of Physics and Astronomy, Michigan State University, East Lansing MI, U.S.A.), Eric M. Patrick (Department of Physics and Astronomy, Michigan State University, East Lansing MI, U.S.A.), Andrea A. Putnam (Department of Biochemistry, Center of RNA Molecular Biology, Case Western Reserve University, Cleveland OH, U.S.A. ), Eckhard Jankowsky (Department of Biochemistry, Center of RNA Molecular Biology, Case Western Reserve 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 DEAD-box family of RNA helicases is the largest of all known RNA helicase families, where they remodel RNA by binding and unwinding duplex RNA via an ATP-dependent mechanism. Lacking translocation capability, these helicases are known to unwind only a limited amount, < 10-20 bp, locally nearby the site of binding. However, some DEAD-box RNA helicases have recently been shown to unwind efficiently upon oligomerization. The DEAD-box RNA helicase Ded1p has been observed to efficiently unwind RNA duplexes possessing a single-stranded RNA (ssRNA) tail in a hypothesized trimer structure. The detailed mechanism of Ded1p assembly and unwinding of RNA remains unknown. We have used combined high-resolution tweezers and single-molecule fluorescence spectroscopy in order to directly observe the detailed step-by-step binding and dissociation of Ded1p protomers and subsequent unwinding and rezipping of duplex RNA. The overall trimer-like activity previously observed in ensemble experiments was recapitulated. A complete general model of unwinding is developed whereby unwinding is initiated by binding of a single Ded1p to the duplex adjacent ssRNA tail. Subsequently, in sequence two additional Ded1p bind and unwind a 5-7 bp portion of the duplex each resulting in the full 16 bp duplex unwinding. The reaction is highly dynamic and stochastic, with unwinding combining with frequent rezipping reversals. Rezipping reversals are suppressed when ATP hydrolysis is suppressed via use of an ATP analog. While unwinding and rezipping are easily captured by high-resolution tweezers, Ded1p binding and dissociation on ssRNA tail was measured via the protein-induced-fluorescence-enhancement (PIFE) signal from the fluorophore-labeled tail. The combined methods allowed us to fully observe the coordinated binding and unwinding of the RNA substrate by three combined individual protomers of the Ded1p.
Keywords: RNA helicases, Optical Tweezers, Single Molecule