Limited Proteolysis Analysis of a DEAD‐box Protein and its Domain Truncated Variants

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RNA helicases play a crucial role in virtually all aspects of RNA metabolism, and although they share a highly conserved structure, the enzymes exhibit a wide variety of biochemical activity. One family of RNA helicases is DEAD‐box proteins, and while their functions are diverse, the one defining activity of these proteins is their ability to hydrolyze ATP in the presence of single‐stranded or double‐stranded RNA.1 ATPase activity leads to changes in RNA affinity, enabling ligand dissociation and cyclic rebinding to promote correct RNA folding in the cell. For example, Rok1p is a yeast DEAD‐box protein essential in ribosomal RNA folding, and is characterized by a catalytic core where ATP and RNA bind, as well as two peripheral domains.2 The peripheral domains (N‐terminal domain or NTD, and the C‐terminal domain, or CTD), are proposed to regulate ATPase activity as well as provide a region for RNA specificity. Thus, peripheral domains could play a role in regulating the overall structure of the protein and its mechanism of RNA refolding. To monitor peripheral effects, the structural changes of Rok1p in the presence and absence of a ligand were analyzed using limited proteolysis under various conditions. These results were subsequently compared to the truncated variant, Rok1p‐ΔCTD. This comparative analysis determined the structural role of the NTD, and to some extent, the CTD. Time dependent proteolysis suggests that the protein is more dynamic at higher temperatures. In the presence of RNA or a nucleotide, the level of structure fluctuation increases. The observed level of conformational changes is minimal yet has dramatic effects on the ATPase activity of the protein. Specifically, the results show that the protein does not occupy a fully extended core state, which accounts for the unique catalytic activity of Rok1p.Support or Funding InformationThe Thomas Lord Charitable Trust FellowshipThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.