Abstract 1758 Kinetic analysis of ATP association of the DEAD-box protein Rok1p and catalytic core

DEAD-box proteins are a large family of ATP-dependent RNA helicases defined by 12 conserved motifs. These proteins are comprised of two independent RecA-like domains that form a catalytic core. DEAD-box proteins have critical roles in most RNA-mediated processes such as transcription, splicing, ribosome biogenesis, mRNA export, translation, and RNA decay. DEAD-box proteins can populate two conformational states: an open-disordered state and a closed-ordered state. ATP hydrolysis is driven and regulated by these open and closed states. For example, Rok1p's initial conformational state controls its RNA dependency. Previous kinetics studies suggest that the initial state is regulated by Rok1p's N-terminal peripheral domain (NTD). Understanding the effects of the peripheral domain and initial conformational states on ATP binding will further the development of DEAD-box inhibitors. Thus, this study aims to observe the temperature dependency of the ATP association rate (kon) with wtRok1p and Rokp's catalytic core. The rate of ATP association was monitored by stop-flow fluorescent spectroscopy utilizing trinitrophenol-ATP (TNP-ATP). The association of ATP and wtRok1p was observed to follow second-order rate kinetics. At low temperatures, the kon was 0.1/μMs. The temperature dependency of kon was nonlinear, suggesting a thermal protein conformational change. In the absence of peripheral domains, the temperature dependency of kon was linear. Using these results, we can present a model in which the peripheral domains can cause a shift in the equilibrium between the open and closed states.