A Conserved Phenylalanine of Motif IV in Superfamily 2 Helicases Is Required for Cooperative, ATP-Dependent Binding of RNA Substrates in DEAD-Box Proteins

Abstract

We have identified a highly conserved phenylalanine in motif IV of the DEAD-box helicases that is important for their enzymatic activities. In vivo analyses of essential proteins in yeast showed that mutants of this residue had severe growth phenotypes. Most of the mutants also were temperature sensitive, which suggested that the mutations altered the conformational stability. Intragenic suppressors of the F405L mutation in yeast Ded1 mapped close to regions of the protein involved in ATP or RNA binding in DEAD-box crystal structures, which implicated a defect at this level. In vitro experiments showed that these mutations affected ATP binding and hydrolysis as well as strand displacement activity. However, the most pronounced effect was the loss of the ATP-dependent cooperative binding of the RNA substrates. Sequence analyses and an examination of the Protein Data Bank showed that the motif IV phenylalanine is conserved among superfamily 2 helicases. The phenylalanine appears to be an anchor that maintains the rigidity of the RecA-like domain. For DEAD-box proteins, the phenylalanine also aligns a highly conserved arginine of motif VI through van der Waals and cation-interactions, thereby helping to maintain the network of interactions that exist between the different motifs involved in ATP and RNA binding.The putative RNA helicases constitute a ubiquitous group of enzymes that are associated with all of the processes involving RNA, including transcription, ribosome biogenesis, mRNA splicing, RNA export, translation, and RNA degradation (reviewed in references 10, 30, 37, 42, and 46). They are characterized by two linked RecA-like domains (domains 1 and 2) that contain seven to eight conserved motifs (I, Ia, Ib, II, III, IV, V, and VI), and they are closely related to many of the DNA helicases (43). These RNA and DNA helicases are classified into either superfamily 1 (SF1) or superfamily 2 (SF2) according to the characteristics of the conserved motifs (17). Although some DNA and RNA helicases are known to have processive unwinding activity, most putative helicases show no or only weak activity in in vitro tests (46). Because contiguous double-stranded RNA is rare in vivo, RNA helicases may play a role primarily in RNA-RNA or RNA-protein remodeling (20,25,40,48,50) or as place setters to ensure the unidirectionality of complex reactions such as splicing or ribosomal biogenesis (46). Nevertheless, all of these family members are collectively known as helicases in the literature (17). Hence, we will use the term helicase in this paper as a generic term rather than as a functional description.The largest family of RNA helicases is the DEAD-box proteins of SF2 that typically have the sequence Asp-Glu-Ala-Asp (DEAD) in motif II (31). In addition to the eight conserved motifs, they contain three other features that are characteristic of this family: the upstream Q motif, which is involved in adenine recognition (11, 45), a conserved GG between motifs Ia and Ib (31), and a conserved QxxR, where x is any amino acid, bet...

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.…”

“…Structural studies have provided a first glimpse on the individual interactions between the conserved motifs in the absence or presence of ATP and ssRNA substrate (Caruthers et al, 2000;Story et al, 2001;Cheng et al, 2005;Andersen et al, 2006;Bono et al, 2006;Sengoku et al, 2006;Nielsen et al, 2008;Collins et al, 2009;von Moeller et al, 2009). When both substrates are bound, the helicase motifs engage in a complex network with a multitude of cooperative interactions, complicating the assignment of functional contributions for each individual motif in mutagenesis studies (Banroques et al, 2008). The complete interaction network and its role for DEAD box protein activity are thus not yet fully understood.…”

Cofactor-dependent specificity of a DEAD-box protein

“…Interestingly, cofactors are often RNA-binding proteins (19)(20)(21)(22)(23)(24)(25). We and others therefore postulated that these cofactors could also function to provide specificity for DEAD-box and DEAH-box proteins (1, 18).Ded1 and eIF4A, two of the best-studied DEAD-box proteins (19,24,(26)(27)(28)(29)(30)(31)(32)(33)(34)(35)(36)(37)(38), have known binding cofactors: Gle1 and eIF4G, respectively. Because Ded1 and eIF4A both function in translation initiation, a process that requires the recognition of many diverse mRNAs, sequence specificity for these particular DEADbox proteins may be undesirable.…”

“…Ded1 and eIF4A, two of the best-studied DEAD-box proteins (19,24,(26)(27)(28)(29)(30)(31)(32)(33)(34)(35)(36)(37)(38), have known binding cofactors: Gle1 and eIF4G, respectively. Because Ded1 and eIF4A both function in translation initiation, a process that requires the recognition of many diverse mRNAs, sequence specificity for these particular DEADbox proteins may be undesirable.…”

Erratum to: Structure and Mechanisms of SF1 DNA Helicases

“…These motifs function together to drive directional movement along ssDNA or dsDNA. They participate in the communication between nucleic acid and ATP-binding sites [118]. The ability to unwind dsDNA appears to be provided by additional protein domains which do not contain the helicase motifs [48].…”