RNA plays an essential role in several cellular processes.  These macromolecules are often trapped in non-functional conformational states that require processing factors to achieve an active structure.  For example, the ubiquitous DEAD-box family of proteins binds and hydrolyzes ATP through a series of conformational changes.  It is these structural changes that result in the disruption of secondary and/or tertiary RNA contacts during processing. In most cases, the level of ATP hydrolysis enhancement ranges from 2- to 100-fold in the presence of RNA.  This wide variation of rate enhancements suggest that DEAD-box proteins can variably populate a closed-active and open-inactive conformation, under specific conditions. We have thermodynamically analyzed the hydrolysis activity of the DEAD-box proteins YxiN, Dbp3p, and Rok1p in order to understand the initial conformational state preferentially populated by these proteins. The results of our experiments have shown that the calculated entropic and enthalpic contributions for both YxiN and Dbp3p are nearly identical.  However, the hydrolysis rates of Rok1p produced nonlinear Erying plots suggesting a conformational shift in the protein that results in low RNA-dependent enhancements in activity.  In order to further understand the conformational shift observed in Rok1p, pre-steady state association and dissociation kinetic analyses will be done using stopped-flow fluorescence.

Biochemical analyses of ATP binding and hydrolysis in DEAD-box proteins

249th National Meeting (2015)

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