Visualization of unwinding activity of duplex RNA by DbpA, a DEAD box helicase, at single-molecule resolution by atomic force microscopy

Abstract

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...

Nonspecific binding to structured RNA and preferential unwinding of an exposed helix by the CYT-19 protein, a DEAD-box RNA chaperone

“…2). This result demonstrates that CYT-19 possesses RNA unwinding activity, as expected by analogy with other DExD͞ H-box proteins (10)(11)(12)(13)(14)(15)(16)(17). § An independent assay using the oligonucleotide cleavage activity of the ribozyme gave the same results within error (Fig.…”

“…Although they are homologous to DNA helicases, and several are known to possess helicase activity in vitro (10)(11)(12)(13)(14)(15)(16)(17), rearrangements of structured RNAs may also involve disruptions of tertiary structure or even displacement of bound proteins (18)(19)(20). Studying the mechanisms of DExD͞H-box proteins on their physiological substrates is difficult because the ''starting state'' of the RNA is typically poorly defined, as discrete misfolded species are difficult to populate and trap, and because most of the substrate RNAs do not have simple activities that can be used to distinguish the native state from structurally related misfolded species.…”

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

“…These experiments are not performed under equilibrium conditions, but nevertheless they have been very valuable for comparing relative affinities of one DEAD box protein for different nucleotides, or for comparing nucleotide or RNA affinities of different DEAD box protein mutants. Nucleotide binding constants have also been derived from 'equilibrium filtration' experiments (Polach and Uhlenbeck, 2002), and RNA binding constants have been determined in electrophoretic mobility shift assays (Pause et al, 1993;Henn et al, 2001;Polach and Uhlenbeck, 2002;Cordin et al, 2004;Karginov et al, 2005;Talavera et al, 2006;Banroques et al, 2008;Liu et al, 2008). In these experiments, the equilibrium is only minimally perturbed.…”

Characterization of the DNA Damage-inducible Helicase DinG from Escherichia coli

“…Not listed in Table III is a helicase-like enzyme UvrB capable of limited strand separation in a complex with the UvrA protein during nucleotide excision repair (35). Two E. coli helicases, DbpA (36,37), involved in ribosome maturation, and RhlB (38), a subunit of the E. coli RNA degradosome, have been shown to possess RNA-RNA unwinding activity. The genes hepA (39), hrpA and rhpB (40), rhlE (41), phoH and yjhR (42), lhr (43), yejH (34), srmB (44), b1808 (34) and deaD (45) have been predicted to encode DNA and RNA helicases, but the corresponding proteins have never been purified, and their helicase activities have yet to be demonstrated.…”