Analysis of DEAD-Box Proteins in mRNA Export

DEAD-box ATPases/helicases are a large family of enzymes (>35 in humans) involved in almost all aspects of RNA metabolism including ribosome biogenesis, RNA splicing, export, translation, and decay. Many members of this family are ATP-dependent RNA-binding proteins that interact with the RNA phosphodiester backbone and promote structural remodeling of target complexes through ATP binding and hydrolysis. Here, we describe the methods used in our laboratory to characterize the DEAD-box ATPase Dbp5 of Saccharomyces cerevisiae. Dbp5 is essential for the process of mRNA export in budding yeast and highly conserved orthologs can be found in all eukaryotes. Specifically, we describe enzyme assays to measure the catalytic activity of Dbp5 in association with RNA and known binding partners, as well as assays developed to measure the binding affinities and release kinetics of RNA and adenosine nucleotides from Dbp5. These assays have provided important information that has shaped our current models of Dbp5 function in mRNA export and should be useful for the characterization of other DEAD-box family members.