The Escherichia coli protein DbpA is unique in its subclass of DEAD box RNA helicases, because it possesses ATPase-specific activity toward the peptidyl transferase center in 23S rRNA. Although its remarkable ATPase activity had been well defined toward various substrates, its RNA helicase activity remained to be characterized. Herein, we show by using biochemical assays and atomic force microscopy that DbpA exhibits ATP-stimulated unwinding activity of RNA duplex regardless of its primary sequence. This work presents an attempt to investigate the action of DEAD box proteins by a single-molecule visualization methodology. Our atomic force microscopy images enabled us to observe directly the unwinding reaction of a DEAD box helicase on long stretches of double-stranded RNA. Specifically, we could differentiate between the binding of DbpA to RNA in the absence of ATP and the formation of a Y-shaped intermediate after its progression through double-stranded RNA in the presence of ATP. Recent studies have questioned the designation of DbpA, in particular, and DEAD box proteins in general as RNA helicases. However, accumulated evidence and the results reported herein suggest that these proteins are indeed helicases that resemble in many aspects the DNA helicases.M any putative RNA helicases are members of the DEAD box protein family, which catalyzes the hydrolysis of ATP in the presence of RNA presumably unwinding the duplex regions of RNA and RNA-DNA hybrids. They are characterized by the ''DEAD'' motif (Asp-Glu-Ala-Asp) as well as by seven other conserved amino acid motifs including two ATP binding domains A and B (1-3). These proteins are found in a wide range of organisms ranging from viruses to higher eukaryotes. Importantly, RNA helicases participate in many essential cellular processes such as transcription, translation, ribosome assembly, cell differentiation, cell development, RNA processing, and mRNA splicing (4-6). Furthermore, the unwinding of the RNA secondary structure is the rate-limiting step in obtaining the functional conformation of RNA, required in these biological processes (2). Therefore, helicases may play a key role in regulating these biological processes by controlling RNA structures. Although the unwinding activity has been demonstrated in vitro for a few RNA helicases (7-11), it has not been shown for many other members of the DEAD box family. It was suggested that, unlike DNA helicases (12), RNA helicases may not be required to unwind long stretches of double-stranded RNA (dsRNA; ref.2). The mode of action of a DEAD box helicase was recently analyzed in detail in the vaccinia nucleoside-triphosphate phosphohydrolase-II (NPH-II) protein (11). NPH-II exhibited highly processive 3Ј to 5Ј helicase activity, in an ATP-dependent manner. These results raised the possibility that the mode of action of RNA and DNA helicases is similar and that the differences between them may be the result of their particular substrates and their interactions with other proteins (10).DbpA was identified by its hom...
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...
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