Poster abstracts
Poster number 143 submitted by Kathryn Sutter
Comparative Analysis of ATP Dissociation from Rok1p Variants of in the presence of RNA
Kathryn Sutter (Biochemistry Allegheny College), Amanda DiLoreto, Lisa Yoder, Zachary Iezzi, and Ivelitza Garci (Biochemistry Allegheny College)
Abstract:
Superfamily 2 helicases (SF2) are ubiquitous and often associated with RNA metabolism. For example, 14 DEAD-box proteins, members of the SF2 family, play a vital role in Saccharomyces cerevisiae ribosome biogenesis. These ATP-dependent unwindases use conformational changes to guide and regulate RNA-RNA, RNA-protein, and/or protein-protein interactions during rRNA processing. Structurally, DEAD-box proteins have 2 Rec-A like domains as well as an N-terminal (NTD) and/or C-terminal (CTD) domains. Peripheral domains are hypothesized to facilitate RNA binding and/or protein scaffolding. To investigate the role of the peripheral domains on ATP binding, variants of Rok1p (Rok1p-ΔCTD, Rok1p-ΔNTD, Rok1p-ΔNTD-ΔCTD) were constructed and biochemically analyzed. Specifically, ATP dissociation rates were measured in the presence of RNA using stopped-flow fluorescence spectroscopy. The dissociation of ATP yielded a bi-exponential regression indicating a two-step mechanism (kfast and kslow). The proposed mechanism involves a conformational change (kslow) before TNP-ATP release (kfast). Measured rates were comparatively analyzed as a function of temperature. This study illustrates that RNA binding, in the presence of NTD, generally increases the enthalpic contribution while decreasing the entropic contribution for kfast. This observation is similar for constructs that lack the NTD when comparing kslow. Interestingly, the presence of the NTD affects the conformation change needed to release TNP-ATP resulting in an enthalpically driven process.
Keywords: DEAD-box protein