Poster abstracts
Poster number 31 submitted by Amanda DiLoreto
Kinetic Analysis of ATP Dissociation from wt-Rok1p and its truncated variants
Amanda DiLoreto (Biochemistry, Allegheny College), Kathryn Sutter (Biochemistry, Allegheny College), Lisa Yoder (Biochemistry, Allegheny College), Zachary Iezzi (Biochemistry, Allegheny College), Ivelitza Garcia (Biochemistry, Allegheny College)
Abstract:
Ribosomal RNA processing is a highly regulated pathway, requiring many trans-acting proteins; for example, 14 DEAD-box proteins play essential roles in rRNA folding. The various functions of DEAD-box proteins are strongly coupled to conformational changes that take place during ATP hydrolysis. Structurally, these proteins are modular, containing two Rec-A like domains that bind ATP and RNA, as well as N-terminal and C-terminal peripheral domains (NTD and CTD). The Saccharomyces cerevisiae DEAD-box protein Rok1p and Rok1p truncated variants were analyzed utilizing stopped-flow fluorescence spectroscopy in the absence of RNA to investigate peripheral domain effects on ATP dissociation. Dissociation rates were determined at various temperatures ranging from 16 °C to 40 °C. TNP-ATP fluorescence quenching as a function of time resulted in a double exponential decay curve, indicating a two-step dissociation mechanism (kfast and kslow). The slow dissociation step suggests that a conformational change is required prior to TNP-ATP release. A thermodynamic-comparative analysis of Rok1p truncation variants showed variable peripheral domain effects on nucleotide dissociation. In the presence of the CTD, the slow conformational change is entropically driven while the release of TNP-ATP is enthalpic. Protein constructs containing the NTD exhibited a gradual inversion in thermodynamic contributions. This study illustrates that both the NTD and CTD affect the ATP binding pocket.
Keywords: dead box protein