DEAD-box proteins are a family of RNA helicases important in most RNA folding and processing pathways. This essential family of proteins utilizes the conformational changes associated with ATP hydrolysis to drive RNA folding in the cell. DEAD-box proteins contain 2 RecA-like domains and 12 conserved motifs. In Saccharomyces cerevisiae, 15 out of 25 DEAD-box proteins are dedicated to ribosome biogenesis. For example, the DEAD-box protein Rok1p is essential for pre-rRNA folding and cleavage of ITS1 during ribosome biogenesis. The Rok1p protein utilizes ATP hydrolysis conformation changes for regulation, modulation of RNA structures, and modulation of Rrp5 complexes. Specifically, this DEAD-box protein has been observed to toggle between open-disordered and closed-order states to regulate its activity and recruitment within the cell. Thus, nucleotide association rates for Rok1p in the absence of RNA were determined using stopped-flow fluorescence spectroscopy and global fitting. Thermodynamic parameters derived from the second-order association rate constant are temperature-dependent and consistent with a structural thermal shift. This experimental model mirrors the hypothesized regulation pathway for Rok1p. Achieving regulation through conformational tuning of an active site imposes important considerations toward the design of active site inhibitors.

Kinetic analysis of ATP associated in the DEAD-box protein Rok1p

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