Abstract
ATP-dependent DEAD-box helicases constitute one of the largest families of RNA helicases and are important regulators of most RNA-dependent cellular processes. The functional core of these enzymes consists of two RecA-like domains. Changes in the interdomain orientation of these domains upon ATP and RNA binding result in the unwinding of double-stranded RNA. The DEAD-box helicase DbpA from E. coli is involved in ribosome maturation. It possesses a C-terminal RNA recognition motif (RRM) in addition to the canonical RecA-like domains. The RRM recruits DbpA to nascent ribosomes by binding to hairpin 92 of the 23S rRNA. To follow the conformational changes of Dbpa during the catalytic cycle we initiated solution state NMR studies. We use a divide and conquer approach to obtain an almost complete resonance assignment of the isoleucine, leucine, valine, methionine and alanine methyl group signals of full length DbpA (49 kDa). In addition, we also report the backbone resonance assignments of two fragments of DbpA that were used in the course of the methyl group assignment. These assignments are the first step towards a better understanding of the molecular mechanism behind the ATP-dependent RNA unwinding process catalyzed by DEAD-box helicases.
Highlights
DEAD box helicases constitute the largest family of RNA helicases in eukaryotes and are found in all organisms (Fairman-Williams et al 2010)
DEAD box helicases consist of two RecA-like domains (Fig. 1) and use the energy generated by ATP hydrolysis to unwind short stretches of duplex RNA in a nonprocessive manner (Fairman-Williams et al 2010)
Based on single molecule FRET experiments (Theissen et al 2008) and crystal structures of different DEAD-box helicases with wildly different interdomain orientations (e.g. (Caruthers et al 2000; Story et al 2001; Cheng et al 2005)), it is generally assumed that the two RecA domains tumble independently in the apo state (Linder and Jankowsky 2011)
Summary
DEAD box helicases constitute the largest family of RNA helicases in eukaryotes and are found in all organisms (Fairman-Williams et al 2010). Simultaneous binding of ATP and RNA induces the formation of a closed state, where ATP is buried between the two RecA domains and a bipartite RNA binding site is formed This leads to the destabilization and thereby the unwinding of the RNA duplex (Putnam and Jankowsky 2013). We report here the ILMVA methyl group assignment of full length DbpA These assignments will serve as the basis for further studies on the mechanism behind the allosteric activation of DbpA and on the RNA unwinding mechanism. In this regard, it is noteworthy that neither the isolated C-terminal RecA domain nor the isolated RRM could be obtained in soluble form after expression in E. coli. A direct interaction between the RRM and the C-terminal RecA domain seems likely and might be the basis for the allosteric activation of the helicase activity upon binding of hairpin 92 to the RRM
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