Structural basis for RNA-duplex recognition and unwinding by the DEAD-box helicase Mss116p - PubMed (original) (raw)

Structural basis for RNA-duplex recognition and unwinding by the DEAD-box helicase Mss116p

Anna L Mallam et al. Nature. 2012.

Abstract

DEAD-box proteins are the largest family of nucleic acid helicases, and are crucial to RNA metabolism throughout all domains of life. They contain a conserved 'helicase core' of two RecA-like domains (domains (D)1 and D2), which uses ATP to catalyse the unwinding of short RNA duplexes by non-processive, local strand separation. This mode of action differs from that of translocating helicases and allows DEAD-box proteins to remodel large RNAs and RNA-protein complexes without globally disrupting RNA structure. However, the structural basis for this distinctive mode of RNA unwinding remains unclear. Here, structural, biochemical and genetic analyses of the yeast DEAD-box protein Mss116p indicate that the helicase core domains have modular functions that enable a novel mechanism for RNA-duplex recognition and unwinding. By investigating D1 and D2 individually and together, we find that D1 acts as an ATP-binding domain and D2 functions as an RNA-duplex recognition domain. D2 contains a nucleic-acid-binding pocket that is formed by conserved DEAD-box protein sequence motifs and accommodates A-form but not B-form duplexes, providing a basis for RNA substrate specificity. Upon a conformational change in which the two core domains join to form a 'closed state' with an ATPase active site, conserved motifs in D1 promote the unwinding of duplex substrates bound to D2 by excluding one RNA strand and bending the other. Our results provide a comprehensive structural model for how DEAD-box proteins recognize and unwind RNA duplexes. This model explains key features of DEAD-box protein function and affords a new perspective on how the evolutionarily related cores of other RNA and DNA helicases diverged to use different mechanisms.

PubMed Disclaimer

Figures

Figure 1

Figure 1. The distinct substrate binding characteristics of the helicase core domains of Mss116p

a, Schematic of the domain architecture of the helicase core of Mss116p (D1, blue; D2, green; C-terminal extension of D2 (CTE), orange), indicating conserved DEAD-box sequence motifs defined according to Fairman-Williams et al. (2010). Full-length Mss116p contains additional unstructured N-terminal (residues 37–87) and C-terminal (residues 598–664) regions that are not required for helicase activity,. b, Affinity of ATP for D1, D2, and the full helicase core (D1D2) measured by gel filtration chromatography under equilibrium conditions. ATP binding was also assessed by an ATP-agarose binding assay (Supplementary Fig. 1). c, Affinity of FAM-dsRNA (Fig. 2a) for MBP-tagged D1, D2 and D1D2 determined by fluorescence anisotropy measurements. Similar results for dsRNA binding were obtained by EMSA (Supplementary Fig. 2). Error bars in b and c represent the standard error for at least three independent measurements, and the error in the _K_d represents the standard error of the non-linear regression (see Full Methods). NB indicates no significant binding.

Figure 2

Figure 2. Crystal structures of Mss116p D2 bound to A-form duplexes

a, The 14-bp self-complementary GC-rich RNA duplex substrate. b, The 14-bp GC-rich chimeric RNA-DNA duplex substrate. c-e, Orthogonal views of the D2-dsRNA complex colored as in Fig. 1a and Fig. 2a. Helix α 14 of D2, which contains motif IVa, faces the major groove of the dsRNA, and α 18 and α 20 of the CTE face the minor groove of the dsRNA. f-h, Orthogonal views of the D2-dsRNA-DNA complex, colored as in Fig. 1a and Fig. 2b, in which D2 is bound to two stacked 14-bp chimeric RNA-DNA duplexes.

Figure 3

Figure 3. Interactions between Mss116p D2 and duplex RNA

a, Schematic of RNA-protein interactions observed in the D2-dsRNA structure. The dsRNA interacts with conserved DEAD-box motifs IV-Va of D2 (green) and the CTE of D2 (orange). The box indicates that the interaction is maintained in closed-state Mss116p. RNA bases are numbered according to their position relative to ssRNA in closed-state Mss116p (see Supplementary Fig. 4d). Similar nucleic acid-protein interactions were observed in the D2-dsRNA-DNA structure (Supplementary Fig. 5). b, Interactions between strand 1 (yellow) of the duplex RNA and D2 (green). c, Interactions between duplex RNA and the CTE of D2 (orange).

Figure 4

Figure 4. RNA duplex binding and unwinding by Mss116p

a, Model for the modular roles of the helicase core domains of Mss116p during RNA recognition and unwinding. Although ATP and duplex RNA could bind in either order, the binding of ATP to D1 is shown as a first step because Mss116p-ATP complexes are likely pre-populated at physiological concentrations of ATP (>1 mM). b. Comparison of the position and interactions of the flexible motif Va loop (residues 435–440) in the D2-dsRNA and closed-state structures of Mss116p. The closed-state helicase core of Mss116p (PDB = 3I5X) bound to ssRNA (U10-RNA; yellow) and adenosine nucleotide (AMP-PNP; black) is shown with domains colored as in Fig. 1a. In the D2-dsRNA structure, the motif Va loop (green) interacts with strand 1 of dsRNA (yellow), whereas in the closed-state structure, the loop (red) shifts to a different position where motif Va helps to form the ATP-binding site. c, Surface representation of closed-state Mss116p with N1–N10 of U10-RNA (yellow) indicated. d, Surface representation of closed-state Mss116p with dsRNA modeled in the duplex RNA-binding pocket of D2. Sterically incompatible regions of D1 are highlighted in red. e, Change in trajectory of strand 1 and predicted displacement of strand 2 of bound dsRNA by D1 upon core closure of Mss116p with arrows indicating regions of the substrate that are displaced.

Similar articles

Cited by

References

    1. Linder P, Jankowsky E. From unwinding to clamping - the DEAD box RNA helicase family. Nat Rev Mol Cell Biol. 2011;12:505–516. - PubMed
    1. Jarmoskaite I, Russell R. DEAD box proteins as RNA helicases and chaperones. WIREs: RNA. 2011;2:135–152. - PMC - PubMed
    1. Yang Q, Del Campo M, Lambowitz AM, Jankowsky E. DEAD-box proteins unwind duplexes by local strand separation. Mol Cell. 2007;28:253–263. - PubMed
    1. Pan C, Russell R. Roles of DEAD-box proteins in RNA and RNP folding. RNA Biol. 2010;7:667–676. - PMC - PubMed
    1. Huang HR, et al. The splicing of yeast mitochondrial group I and group II introns requires a DEAD-box protein with RNA chaperone function. Proc Natl Acad Sci USA. 2005;102:163–168. - PMC - PubMed

Publication types

MeSH terms

Substances

Grants and funding

LinkOut - more resources