Poster abstracts

Poster number 27 submitted by Huijue Jia

RNA duplex unwinding by the DEAD-box protein Ded1p involves multiple enzyme units that utilize ATP differentially

Huijue Jia (Department of Biochemistry, and Center for RNA Molecular Biology, Case Western Reserve University, Cleveland, OH), Fei Liu (Department of Physics, Case Western Reserve University, Cleveland, OH), Quansheng Yang (Department of Biochemistry, and Center for RNA Molecular Biology, Case Western Reserve University, Cleveland, OH), Eckhard Jankowsky (Department of Biochemistry, Center for RNA Molecular Biology, and Department of Physics, Case Western Reserve University, Cleveland, OH)

Abstract:
In virtually all aspects of RNA metabolism, DEAD-box proteins carry out a range of ATP-dependent RNA and RNP remodeling reactions, including unwinding of RNA duplexes. Duplex unwinding by DEAD-box proteins differs from the translocation-based mechanism employed by structurally related canonical DNA and viral RNA helicases. Local strand separation, rather than translocation, underlies the unwinding activity of DEAD-box proteins. In this unwinding mode the enzyme is first loaded directly onto the duplex by a single stranded substrate region. Subsequently, the enzyme actively opens only part of the duplex locally around its binding site, which is sufficient to promote the rapid separation of duplexes shorter than approximately one and a half helical turns.

To understand this novel helicase mechanism in more detail, we have dissected individual steps of the strand separation process for the DEAD-box protein Ded1p from Saccharomyces cerevisiae. Here we show that the single stranded substrate region serves solely to load the enzyme on the duplex, with no effect on the actual unwinding. We further show that multiple protomers of Ded1p bind the single stranded substrate region and that binding site size for individual Ded1p protomer comprises approximately 10 nucleotides. Finally, we demonstrate that Ded1p units on the single stranded substrate region do not directly participate in the unwinding process but serve to load additional Ded1p units onto the duplex region, and that only ATP hydrolysis from Ded1p units binding directly to the duplex is required for unwinding. Our data reveal a complex unwinding process for the DEAD-box protein Ded1p involving multiple enzyme units, which utilize ATP differentially. This intricate unwinding mechanism may be advantageous for accommodating the many distinct RNA substrates that Ded1p may encounter during its various cellular functions.

Keywords: DEAD-box protein, RNA duplex unwinding, oligomerization