YxiN Is a Modular Protein Combining a DExD/H Core and a Specific RNA-binding Domain

Abstract

DEx Members of the DExD / H helicase family participate in many cellular processes involving rearrangement of RNA-RNA and RNA-protein interactions (1, 2). DEx D / H proteins contain a conserved catalytic core consisting of two RecA-like domains that bind ATP and single-stranded RNA (3). Similar to other RecA-like ATPases (4), the cycle of ATP binding, hydrolysis, and release is coupled to a conformational change in the core. In DEx D / H proteins, this results in translocation of the protein along single-stranded RNA and subsequent duplex destabilization and/or ribonucleoprotein disruption (5, 6). Outside of the catalytic core, DEx D / H proteins often contain ancillary N-and C-terminal extensions that correlate with their specific cellular functions. In many cases, helicases are part of multiprotein complexes, and the putative role of the ancillary domains is to recruit the helicase to the complex by proteinprotein interactions (7). Alternatively, the ancillary domains can directly bind to a specific RNA substrate, delivering the helicase to its site of action.Bacillus subtilis DEx D / H protein YxiN and Escherichia coli DbpA define a group of bacterial orthologs with a conserved C-terminal domain of ϳ80 amino acids (Fig. 1A). Both YxiN and DbpA specifically recognize the A-site region of 23 S rRNA, including hairpin 92, suggesting that the RNA specificity is conferred by the C-terminal domain (8 -11). In a domain swap experiment, the C-terminal domain of YxiN was appended to the catalytic core of SrmB, a non-sequence-specific RNA helicase from E. coli (12). The resulting chimera possessed the RNA specificity of YxiN but had catalytic properties similar to those of the parental helicase, SrmB. These results indicate that the C-terminal domain of YxiN is capable of imparting specificity in the context of another DEx D / H box protein, suggesting that the sequence-specific RNA binding and helicase activities are separable and that this group of DEx D / H proteins may be functionally modular. To prove this hypothesis, the helicase and C-terminal domains of YxiN were prepared here as separate polypeptides. This approach permits the intrinsic activities of the catalytic core and the C-terminal domain to be assayed independently and compared with the full-length protein. EXPERIMENTAL PROCEDURES Plasmids for Expression of Recombinant YxiN Fragments-Codingsequences for YxiN protein fragments were PCR-amplified from the native B. subtilis sequence in an existing plasmid (11) and cloned by restriction/ligation into the pTWIN1 vector of an intein-based expression system (New England Biolabs) using the unique NdeI and SpeI sites of the vector. Primers were designed such that the subcloned fragments deleted the first of the tandem inteins of the parent vector and placed the C-terminal residue of each encoded YxiN protein fragment flush with the N-terminal residue of the second intein. In this manner, no extraneous residues remained at the C terminus of the target after selfcleavage of the target-intein fusion. The plasmids ...

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

“…The K d values for singlestranded RNA are in the low micromolar range, and no or weak binding of dsRNA has been detected (Lorsch and Herschlag, 1998a). In agreement with common properties of the helicase core, the truncation of flanking domains in DEAD box helicases reduces their RNA affinity to the level observed for eIF4A (Karginov et al, 2005;Grohman et al, 2007;Mohr et al, 2008).…”

“…Instead, conserved lysines in two loops might be involved in RNA binding. The isolated RBD binds hairpin 92 with similar affinity as the full-length YxiN protein (Karginov et al, 2005;Wang et al, 2007). YxiN unwinds a short double helix adjacent to hairpin 92, and it has been suggested that the RBD functions as an anchor to direct the unwinding activity of the helicase core to this region.…”

“…For example, the C-terminal domains of the splicing helicases CYT-19 and Mss116 mediates interactions with structured RNA (Grohman et al, 2007;Mohr et al, 2008). In contrast, the C-terminal domains of DbpA and YxiN specifically interact with a hairpin in ribosomal RNA (Diges and Uhlenbeck, 2001;Tsu et al, 2001;Kossen et al, 2002;Karginov et al, 2005;Wang et al, 2006), and the C-terminal domain of Hera provides high affinity for ribosomal RNA and RNase P RNA, among others (Morlang et al, 1999;Linden et al, 2008). The influence of domains flanking the helicase core on RNA binding and on DEAD box mechanism in general will be discussed in detail in 'Modulation of the helicase core activity by interacting partners and flanking domains'.…”

“…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.…”

Unwinding RNA's secrets: advances in the biology, physics, and modeling of complex RNAs

“…Although the details of these processes remain elusive, it is plausible that DExD/H-box proteins capture the energy of ATP hydrolysis to drive the transient formation of thermodynamically unstable states that are part of the normal reaction cycle. Although non-specific DEAD-box chaperones could be important for these processes, presumably most of the required structural changes are mediated by dedicated DExD/H-box proteins, which use specific RNA or protein interactions to position themselves within complexes for rearrangement of the desired structural element [47][48][49].…”