Solution structures of DEAD-box RNA chaperones reveal conformational changes and nucleic acid tethering by a basic tail

Abstract

The mitochondrial DEAD-box proteins Mss116p of Saccharomyces cerevisiae and CYT-19 of Neurospora crassa are ATP-dependent helicases that function as general RNA chaperones. The helicase core of each protein precedes a C-terminal extension and a basic tail, whose structural role is unclear. Here we used small-angle X-ray scattering to obtain solution structures of the full-length proteins and a series of deletion mutants. We find that the two core domains have a preferred relative orientation in the open state without substrates, and we visualize the transition to a compact closed state upon binding RNA and adenosine nucleotide. An analysis of complexes with large chimeric oligonucleotides shows that the basic tails of both proteins are attached flexibly, enabling them to bind rigid duplex DNA segments extending from the core in different directions. Our results indicate that the basic tails of DEAD-box proteins contribute to RNA-chaperone activity by binding nonspecifically to large RNA substrates and flexibly tethering the core for the unwinding of neighboring duplexes.EAD-box proteins comprise the largest family of helicases and play critical roles in all aspects of RNA metabolism, including RNA splicing, translation, ribosome assembly, RNA degradation, and RNA transport (1, 2). Although their functions vary broadly, all DEAD-box proteins have a conserved helicase core consisting of two RecA-like domains (denoted domains 1 and 2) separated by a flexible linker and operate by a mechanism involving conformational changes within this core (3-6). These conformational changes couple cycles of ATP binding and hydrolysis to RNA binding and release by the core, enabling DEADbox proteins to promote local RNA unwinding and remodeling of structured RNAs and RNA-protein complexes (3-5).Structural and biochemical studies have given important insights into how DEAD-box proteins unwind RNA (2-4). The two helicase core domains are separated from each other in an open state in the absence of substrates, but they interact to form a compact, closed state upon binding of ATP and RNA. In this closed state, the interface between the two core domains forms a catalytic site for ATP hydrolysis and an RNA-binding cleft, which can accommodate a short region of a duplex strand (7). The tight binding of the RNA strand within this cleft results in a bend that is incompatible with partner-strand base pairing, leading to local RNA unwinding (7-10). ATP hydrolysis and dissociation of the P i product are then necessary to release the bound single-stranded RNA (9,11,12).Whereas the helicase core is central to the biochemical activities of DEAD-box proteins, most also have substantial extensions or additional domains at their N-and/or C-termini (1, 3). These extensions vary widely in size, composition, and function, but many are thought to interact with RNA or protein components of complexes to direct the DEAD-box proteins to desired sites of action. A common type of extension is a basic C-terminal sequence (C-tail), which is typically predicted...

When core competence is not enough: functional interplay of the DEAD-box helicase core with ancillary domains and auxiliary factors in RNA binding and unwinding

“…Instead, they are consistent with favored conformations of the C-tail close to RecA_C and the CTE, in a position to contact the RNA extending from the helicase core (Mallam et al, 2011). Overall, the C-tail appears to serve as a functional tether that interacts electrostatically with exposed helices (Pan et al, 2014) and anchors Mss116 on its large RNA substrate (Mallam et al, 2011). The helicase core then unwinds duplexes within reach from this anchoring site.…”

“…The basic C-tail following the CTE extends outward from RecA_C/CTE, and thus away from the helicase core. The C-tail is flexible, and can cover a large conformational space relative to the helicase core (Mallam et al, 2011). Despite its lack of regular structure, SAXS data argue against a fully extended conformation of the C-tail.…”

“…Following the structured CTE, Mss116 contains an unstructured, basic C-terminal tail (C-tail) in a position to interact with RNA regions further upstream. The C-tails of Mss116 and of the closely related Neurospora crassa DEAD-box protein Cyt-19 have been implicated in binding to structured RNAs (Tijerina et al, 2006;Grohman et al, 2007;Mallam et al, 2011).…”

“…The only structural information on RNA binding by fulllength DEAD-box proteins, including N-and C-terminal regions flanking the helicase core, stems from SAXS experiments on S. cerevisiae Mss116 and Neurospora crassa Cyt 19 (Mallam et al, 2011). The structural model of full-length Mss116 is consistent with the NTE and the basic C-tail being unstructured extensions.…”

“…A large variety of juxtapositions of the two RecA domains has been captured in crystal structures of different DEAD-box helicases cores (Caruthers et al, 2000;Story et al, 2001;Cheng et al, 2005;Andersen et al, 2006;Hogbom et al, 2007;Klostermeier 2013;Mathys et al, 2014;Mohlmann et al, 2014). By contrast, solution studies support a more homogenous, less widely open conformation (Wang et al, 2007;Theissen et al, 2008;Hilbert et al, 2011;Mallam et al, 2011). Binding of ATP and RNA to DEADbox proteins is accompanied by a conformational change of the helicase core to the closed state (Sengoku et al, 2006;Theissen et al, 2008;Aregger and Klostermeier, 2009;Andreou and Klostermeier, 2014;Harms et al, 2014).…”