Assignment of the Ile, Leu, Val, Met and Ala methyl group resonances of the DEAD-box RNA helicase DbpA from E. coli
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ARTICLE
Assignment of the Ile, Leu, Val, Met and Ala methyl group resonances of the DEAD‑box RNA helicase DbpA from E. coli Jan Philip Wurm1 Received: 28 September 2020 / Accepted: 24 November 2020 © The Author(s) 2020
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. Keywords DEAD-box helicase · Ribosome assembly · RNA · Methyl group assignment
Biological context DEAD box helicases constitute the largest family of RNA helicases in eukaryotes and are found in all organisms (Fairman-Williams et al. 2010). They are key players in virtually every step of RNA biology and are implicated in infection and disease (Steimer and Klostermeier 2012). 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 (up to ~ 15–20 nt) in a nonprocessive manner (Fairman-Williams et al. 2010). The residues that are involved in ATP or RNA binding and the allosteric coupling between these two binding sites are highly conserved among all members of the enzyme family (Linder and Jankowsky 2011). The RNA unwinding activity is generally not sequence-specific, but DEAD box helicases usually possess flanking N- and C-terminal sequences that allow their selective recruitment to different cellular * Jan Philip Wurm jan‑[email protected] 1
Department of Biophysics I, University of Regensburg, 93053 Regensburg, Germany
target sites (Fairman-Williams et al. 2010). Examples are sequence-specific RNA binding domains (Hardin et al. 2010) or short unstructured sequence motifs that are used to recruit helicases to their target site via protein–protein interactions (Sharma and Jankowsky 2014). The unwinding mechanism of DEAD-box helicases has been extensively studied and is based on conformational changes between the RecA-like domains, which are connected by a fle
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