Abstract 2178 Understanding DEAD-Box RNA Helicase Regulation by Protein Cofactors and Metabolic Intermediates

Many RNA folding pathways depend on several protein factors to achieve an active native state. These factors belong to different protein families, perform essential roles, and are highly regulated. In yeast, ribosome biogenesis requires hundreds of protein cofactors such as helicases, GTPases, chaperones, scaffolders, nucleases, and other proteins. DEAD-box proteins belong to the largest helicase family, superfamily 2 (SF2). These proteins are defined by 12 conserved motifs, two Rec-A like domains, and RNA-dependent ATP unwindase activity. The yeast DEAD-box protein Rok1p, for example, is essential in the folding and processing of the 18S rRNA. Rok1p is recruited to the pre-rRNA by the protein scaffolder Rrp5. Rrp5 is defined by 12 S1 RNA binding domains and a TPR region that mediates protein recruitment. Previous studies suggest that Rrp5 activates Rok1p by interacting with the N-terminal domain. In this study, the rate of ATP hydrolysis mediated by Rok1p was monitored under various conditions to understand its regulation. The rate of ATP hydrolysis for Rok1p was determined to be 23 ± 1 MPO4/(minMRok1p), irrespective of RNA. In the presence of Rrp5, the rate of ATP hydrolysis for Rok1p was determined to be 20 ± 1 MPO4/(minMRok1p) and 5.5 ± 0.2 MPO4/(minMRok1p) in the presence and absence of RNA, respectively. Thus, Rrp5 activates the RNA dependency of Rok1p. Additionally, this study investigated the effect of known metabolic regulators on ATP hydrolysis. In the presence of Rrp5, Rok1p became less sensitive to the noncompetitive inhibitor ADP. Thus, the tunability of metabolic regulation of Rok1p depends on the availability of variable allosteric sites controlled by Rrp5 binding.