Mechanism of Mss116 ATPase Reveals Functional Diversity of DEAD-Box Proteins

Abstract

Mss116 is a Saccharomyces cerevisiae mitochondrial DEAD-box RNA helicase protein essential for efficient in vivo splicing of all group I and II introns and activation of mRNA translation. Catalysis of intron splicing by Mss116 is coupled to its ATPase activity. Knowledge of the kinetic pathway(s) and biochemical intermediates populated during RNA-stimulated Mss116 ATPase is fundamental for defining how Mss116 ATP utilization is linked to in vivo function. We therefore measured the rate and equilibrium constants underlying Mss116 ATP utilization and nucleotide-linked RNA binding. RNA accelerates the Mss116 steady-state ATPase ~7-fold by promoting rate-limiting ATP hydrolysis, such that Pi release becomes (partially) rate-limiting. RNA binding displays strong thermodynamic coupling to the chemical states of the Mss116-bound nucleotide such that Mss116 with bound ADP-Pi binds RNA more strongly than with bound ADP or in the absence of nucleotide. The predominant biochemical intermediate populated during in vivo steady-state cycling is the strong RNA binding, Mss116-ADP-Pi state. Strong RNA binding allows Mss116 to fulfill its biological role in stabilization of group II intron folding intermediates. ATPase cycling allows for transient population of the weak RNA binding, ADP state of Mss116 and linked dissociation from RNA, which is required for the final stages of intron folding. In cases where Mss116 functions as a helicase, the data collectively favor a model in which ATP hydrolysis promotes a weak-to-strong RNA binding transition that disrupts stable RNA duplexes. The subsequent strong-to-weak RNA binding transition associated with Pi release dissociates RNA-Mss116 complexes, regenerating free Mss116.

Mutational analysis of the functional sites in porcine reproductive and respiratory syndrome virus non-structural protein 10

“…Recently, some studies have found that ATPindependent duplex unwinding can be mediated by DEAD-box proteins, but with lower efficiency than ATPdependent unwinding (Cao et al, 2011;Henn et al, 2010). The binding of ATP to RNA helicase might change the protein's conformation and enhance RNA-protein binding (Henn et al, 2008).…”

DEAD-box protein CYT-19 is activated by exposed helices in a group I intron RNA

“…Binding of ATP and doublestranded RNA to domains 1 and 2, respectively, induces domain closure, which completes the formation of an ATPase active site at the domain interface and introduces steric clashes in the RNA binding site, leading to the displacement of one of the RNA strands (6,7). ATP hydrolysis and inorganic phosphate release are then thought to regenerate the open enzyme conformation (4,8,13). Unlike conventional helicases, DEAD-box proteins have not been found to translocate, limiting the unwinding activity to short helices that can be disrupted in a single cycle of ATP binding and hydrolysis (4,8,9,(14)(15)(16)).…”

Solution structures of DEAD-box RNA chaperones reveal conformational changes and nucleic acid tethering by a basic tail

“…The tight binding of the RNA strand within this cleft results in a bend that is incompatible with partner-strand base pairing, leading to local RNA unwinding (7-10). ATP hydrolysis and dissociation of the P i product are then necessary to release the bound single-stranded RNA (9,11,12).Whereas the helicase core is central to the biochemical activities of DEAD-box proteins, most also have substantial extensions or additional domains at their N-and/or C-termini (1, 3). These extensions vary widely in size, composition, and function, but many are thought to interact with RNA or protein components of complexes to direct the DEAD-box proteins to desired sites of action.…”

“…The tight binding of the RNA strand within this cleft results in a bend that is incompatible with partner-strand base pairing, leading to local RNA unwinding (7-10). ATP hydrolysis and dissociation of the P i product are then necessary to release the bound single-stranded RNA (9,11,12).…”