The "DEAD box" protein DbpA interacts specifically with the peptidyltransferase center in 23S rRNA.

Abstract

The Escherichia coli DEAD (Asp-Glu-AlaAsp) box protein DbpA is a putative RNA helicase and established RNA-dependent ATPase and is the only member of the DEAD box protein family for which a specific RNA substrate, bacterial 23S rRNA, has been identified. We have investigated the nature of this specificity in depth and have localized by deletion mutagenesis and PCR a single region of 93 bases (bases 2496-2588) in 23S rRNA that is both necessary and sufficient for complete activation of ATPase activity of DbpA. This target region forms part of the peptidyltransferase center and includes many bases involved in interaction with the 3' terminal adenosines of both A-and P-site tRNAs. Deletion of stem loops within the 93-base segment abolished ATPase activation. Similarly, point mutations that disrupt base pairing within stem structures ablated stimulation of ATPase activity. These data are consistent with roles for DbpA either in establishing and/or maintaining the correct threedimensional structure of the peptidyltransferase center in 23S rRNA during ribosome assembly or in the peptidyltransferase reaction.DbpA is a member of the large "DEAD box" family of putative RNA helicases, so called because they share the highly conserved motif Asp-Glu-Ala-Asp together with several other conserved functional elements (1-4). DEAD box proteins are found in a wide range of organisms, ranging from bacteria to mammals, and are involved in a variety of processes such as ribosome assembly, initiation of translation, mRNA splicing, and development (2, 3). Multiple DEAD box proteins have been identified in organisms with small genomes [e.g., 20 in yeast (5) and 5 in Escherichia coli (6)], suggesting that they have specific functions that require interaction with specific RNA substrates. Individual members of the DEAD box family are highly conserved, and many are required for viability (7), implying that they perform essential functions in the cell. These proteins are thought to be involved in the local unwinding of RNA secondary structures in a variety of biological processes and may, therefore, play a role in maintaining the optimal conformation of these RNA molecules or in regulating the biological events in which they are involved through controlling RNA structure.The E. coli dbpA gene was originally isolated by hybridization with a probe from the Saccharomyces pombe homolog of p68, one of the prototypic DEAD box proteins (8). However, DbpA is no more related to p68 than to any other members of the DEAD box family. To date, five DEAD box protein genes have been reported in E. coli. Apart from dbpA (8), these include srmB (9) and deaD (10) which, when expressed at high-copy number, will suppress the effect of temperaturesensitive lethal mutations in L24 and S2, respectively, suggesting a role in ribosome biogenesis. Another gene, rhlB, appears essential only in some genetic backgrounds (6), but conditionalThe publication costs of this article were defrayed in part by page charge payment. This articl.-must therefore be hereby ...

The human coronavirus 229E superfamily 1 helicase has RNA and DNA duplex-unwinding activities with 5′-to-3′ polarity

“…The data described here do not allow for definitive conclusions concerning the function of a 59-to-39 helicase activity in coronavirus replication and transcription+ However, because double-stranded replicative intermediates are believed to be the predominant RNA structures in coronaviral RNA synthesis (for a review, see van der Most & Spaan, 1995), it is tempting to speculate that, in analogy to models described for the replisome (reviewed in Baker & Bell, 1998), the coronavirus helicase operates in conjunction with RdRp and, by translocating along the "lagging strand" RNA template, provides the single-stranded RNA template for processive ("leading strand") RNA synthesis+ It is noteworthy that the vaccinia virus NPH-II RNA helicase has recently been shown to be a highly processive enzyme that unwinds long duplex RNA structures (Jankowsky et al+, 2000)+ This data contradicts the previously held belief that processivity can only be attributed to DNA helicases (de la Cruz et al+, 1999) and supports the hypothesis that, at least some viral RNA helicases might be "replicative" helicases in the true sense+ Because coronaviruses are RNA viruses that replicate in the cytoplasm of the infected cell, it appears unlikely that the HEL DNA helicase activity has any relevance to a specific function in viral RNA synthesis+ However, as illustrated for the HCV NS3 helicase, the in vitro DNA helicase activity greatly facilitates biochemical and structural analyses (Preugschat et al+, 1996;Korolev et al+, 1997;Kim et al+, 1998)+ We have used the DNA helicase activity of HEL to investigate the effects of length and sequence variations in the 59 tail of a partial-duplex DNA substrate on duplex unwinding+ The data we have obtained do not allow us to propose a well-defined sequence specificity of the coronavirus helicase, as has been possible for other proteins (FullerPace et al+, 1993;Nicol & Fuller-Pace, 1995;O'Day et al+, 1996;Xu et al+, 1996), but they are a first step in the identification of biologically relevant, coronavirusspecific helicase substrates+…”

DEAD-Box RNA Helicases in Gram-Positive RNA Decay

“…The DbpA protein is famous for its requirement for a specific RNA substrate for efficient ATP hydrolysis (Fuller-Pace et al, 1993). Indeed, this DEAD-box protein is exclusively and heavily stimulated in its activity by the hairpin h92 from the 23S rRNA (Diges and Uhlenbeck, 2001;Nicol and Fuller-Pace, 1995). Recently, it was shown that a dominant-negative mutation in motif VI of dbpA results in a ribosome biogenesis defect (Elles and Uhlenbeck, 2008).…”

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

“…RNA binding domains can provide high affinity, high specificity, or both. The DEAD box proteins DbpA (Escherichia coli) and its B. subtilis homolog YxiN bind to hairpin 92 in the ribosomal 23S rRNA with high affinity and specificity and have been implicated in ribosome biogenesis (FullerPace et al, 1993;Nicol and Fuller-Pace, 1995;Kossen and Uhlenbeck, 1999;Kossen et al, 2002). The crystal structure of the C-terminal RNA-binding domain (RBD) of YxiN adopts a classical RNA-recognition motif (RRM) fold .…”