  DDX3X is a human DEAD-box RNA helicase implicated in many cancers and in
viral progression. In addition to the RecA-like catalytic core, DDX3X contains
N- and C-terminal domains. Here, we investigate the substrate and protein
requirements to support the ATPase activity of a DDX3X construct lacking 80
residues from its C-terminal domain. Our data confirmed previous results that
for an RNA molecule to support the ATPase activity of DDX3X it must contain a
single-stranded-double-stranded region. We investigated protein and RNA
structural reasons for this requirement. First, the RNA substrates consisting
only of a double-helix were unable to support DDX3X binding. A single-stranded
RNA substrate supported DDX3X binding, while an RNA substrate consisting of a
single-stranded-double-stranded region not only supported the binding of DDX3X
to RNA, but also promoted DDX3X trimer formation. Thus, the
single-stranded-double-stranded RNA region is needed for DDX3X trimer
formation, and trimer formation is required for ATPase activity. Interestingly,
the dependence of ATP hydrolysis on the protein concentration suggests that the
DDX3X trimer hydrolyzes only two molecules of ATP. Lastly, a DNA substrate that
contains single-stranded-double-stranded regions does not support the ATPase
activity of DDX3X.
