| DEAD/H box RNA helicases catalyze structural rearrangements in RNA by coupling ATP hydrolysis to the destabilization of RNA helices or ribonucleoprotein complexes. By modulating the structure of RNA, DEAD/H box proteins serve as integral parts of the cellular machinery responsible for all aspects of RNA metabolism. DbpA, a DEAD/H box RNA helicase from E.coli, exhibits its maximal ATPase activity towards a region of the 23S ribosomal RNA known as the peptydyl transferase center. However, the molecular mechanism of substrate recognition and ATPase activity of DbpA is not well understood. As a first step toward understanding how nucleotide binding and hydrolysis are coupled energetically to RNA unwinding, we investigated the mechanism of nucleotide binding to RNA-free DbpA, and characterized the assembly state of DbpA intermediates during the ATPase cycle. Our results indicate that the binding affinities of the nucleotides are dictated primarily by the dissociation rate constant. ATP binds more weakly than ADP when DbpA is in its RNA-free state, suggesting that ATP binding probably occurs when the enzyme is in its RNA-bound state during the productive ATPase cycle. The temperature-dependence of the nucleotide binding kinetics showed that ADP binding occurs with a positive change in the heat capacity, seemingly increasing the conformational flexibility and dynamics of DbpA and suggesting a role of the ADP-bound state during the helicase reaction. Studies of the assembly state of the intermediates strongly argue that DbpA functions as a monomer during its catalytic cycle in vivo. Together, these results provide a preliminary definition of macromolecular interactions in terms of the assembly state, thermodynamics, affinity, and kinetics of the intermediates throughout the ATPase cycle of DbpA. |
