The RNA Helicase DbpA Exhibits a Markedly Different Conformation in the ADP-bound State When Compared with the ATP- or RNA-Bound States

Abstract

The motor enzymes that belong to the family of RNA helicases catalyze the strand separation of duplex RNA via ATP hydrolysis. Among these enzymes, Escherichia coli DbpA is a unique RNA helicase because it possesses ATPase-specific activity toward the peptidyl transferase center in 23 S ribosomal RNA. For this reason, it has been the subject of numerous biochemical and structure-function studies. The ATP-stimulated unwinding activity of DbpA toward specific and nonspecific RNA duplexes has been demonstrated. However, the underlying molecular and structural basis, which facilitates its helicase activities, is presently not known. We combined time-dependent limited proteolysis digestion, fluorescence spectroscopy, and three-dimensional structural homology modeling techniques to study the structural conformations of DbpA with respect to its binding to stoichiometric ratios of RNA and cofactors. We show that the conformational state of DbpA is markedly different in the ADP-bound state than in any other state (ATP-or RNA-bound). These results, together with structural homology studies, suggest that a hinge region located in the core domain of DbpA mediates such conformational changes.RNA adopts specific structures that govern its biological activities in various RNA-dependent cellular processes (1, 2). The correct folding of RNA may be regulated by the ATP-dependent DEXD/H box RNA helicases (3, 4). In this process, the hydrogen bonds of the double-stranded RNA region are disrupted enzymatically by helicases in the course of the unwinding reaction (5, 6). These enzymes are found in a wide range of organisms, ranging from viruses and prokaryotes to lower and higher eukaryotes. RNA helicases participate in many essential cellular processes, such as transcription, translation, ribosome assembly, cell differentiation, cell development, RNA processing, and mRNA splicing (7,8). Therefore, helicases may play a key role in regulating these biological processes by controlling RNA structures.In general, these motor enzymes belong to a large family (designated as superfamily II) characterized by a DEAD/H (Asp-Glu-Ala-Asp/His) box motif in addition to eight other conserved structural motifs in their sequences (Fig. 1) (9, 10). Specifically, the DEAD/H box motif is associated with ATP binding and hydrolysis processes, and therefore, it is thought to affect the unwinding or helicase activity of these enzymes (11,12). Interestingly, all the conserved motifs are positioned in the so-called "core region" of the protein (13). The core region is attached to N-or C-terminal extensions. These N-or C-terminal extensions in RNA helicases provide the enzyme with its substrate specificity and localization, both in the cell and in other protein-protein interactions (5). It was, therefore, proposed that within the helicase superfamily, the core domain scaffold that provides the enzymes with their mechanical activity is structurally and functionally similar (5,14). In addition, it is thought that helicases generally execute their helicase activit...

The mechanism of ATP-dependent RNA unwinding by DEAD box proteins

“…In addition, the lack of contributions of the g-phosphate to nucleotide affinity is an indication for ATP-driven conformational changes. Limited proteolysis experiments provide further evidence for ATP-induced conformational changes (Lorsch and Herschlag, 1998b;Henn et al, 2002;Cheng et al, 2005;Low et al, 2007).…”

“…Indirect evidence for such a conformational reorganization is available from different proteolysis patterns in the presence or absence of substrates (Lorsch and Herschlag, 1998b;Henn et al, 2002;Cheng et al, 2005;Low et al, 2007). Nucleotide binding kinetics support nucleotide-driven conformational rearrangements: ATP and ADP binding follows a single exponential in the absence of RNA, but a second slow phase appears when RNA is present.…”

“…Nucleotide binding can be investigated via fluorescence of mant nucleotides (Henn et al, 2002(Henn et al, , 2008Talavera and De La Cruz, 2005;Karow et al, 2007;Theissen et al, 2008;Karow and Klostermeier, 2009), and RNA binding can be monitored via fluorescence anisotropy using fluorescently labeled RNAs (Karow et al, 2007;Marintchev et al, 2009). Fluorescence spectroscopy has also been employed to directly determine rate constants for nucleotide binding and dissociation (Henn et al, 2002(Henn et al, , 2008 and for RNA unwinding (Karow et al, 2007). The fact that DEAD box proteins are RNA-stimulated ATPases provides a further means to characterize their interactions with RNA and ATP (Lorsch and Herschlag, 1998a,b;Tsu and Uhlenbeck, 1998;Kossen and Uhlenbeck, 1999;Tsu et al, 2001;Polach and Uhlenbeck, 2002;Karow et al, 2007;Henn et al, 2008;Theissen et al, 2008;Karow and Klostermeier, 2009).…”

“…Equilibrium dissociation constants can be obtained directly in fluorescence titrations of fluorescently labeled nucleotides or RNA. Nucleotide binding can be investigated via fluorescence of mant nucleotides (Henn et al, 2002(Henn et al, , 2008Talavera and De La Cruz, 2005;Karow et al, 2007;Theissen et al, 2008;Karow and Klostermeier, 2009), and RNA binding can be monitored via fluorescence anisotropy using fluorescently labeled RNAs (Karow et al, 2007;Marintchev et al, 2009). Fluorescence spectroscopy has also been employed to directly determine rate constants for nucleotide binding and dissociation (Henn et al, 2002(Henn et al, , 2008 and for RNA unwinding (Karow et al, 2007).…”

The Nuclear Pore Complex and Nuclear Transport

“…Such DEAD-box proteins are involved in the alteration of RNA structure, such as the facilitation of RNA duplex formation and unwinding or aiding the association and dissociation of RNA-binding proteins (Jankowsky et al 2001;Fairman et al 2004;Yang and Jankowsky 2006). Much like the GDP/GTP triggered switches for Ran, DEADbox proteins potentially use ADP/ATP for nucleotide-dependent conformational switches (Henn et al 2002;Tran et al 2007b;Henn et al 2008;Fan et al 2009). Dbp5 ATPase activity is activated by Gle1, a protein that exchanges between the nucleus and cytoplasm with a docking site on the cytoplasmic filaments of the NPC (Cole and Scarcelli 2006).…”