tailieunhanh - Báo cáo khoa học: Authentic interdomain communication in an RNA helicase reconstituted by expressed protein ligation of two helicase domains

RNA helicases mediate structural rearrangements of RNA or RNA–pro-tein complexes at the expense of ATP hydrolysis. Members of the DEAD box helicase family consist of two flexibly connected helicase domains. They share nine conserved sequence motifs that are involved in nucleotide binding and hydrolysis, RNA binding, and helicase activity. | ỊFEBS Journal Authentic interdomain communication in an RNA helicase reconstituted by expressed protein ligation of two helicase domains Anne R. Karow Bettina Theissen and Dagmar Klostermeier Department of BiophysicalChemistry University of Basel Switzerland Keywords expressed protein ligation helicase interdomain communication RNA-dependent ATPase Correspondence D. Klostermeier University of Basel Biozentrum Department of Biophysical Chemistry Klingelbergstrasse 70 CH-4056 Basel Switzerland Fax 41 61 267 2189 Tel. 41 61 267 2381 E-mail Received 23 October 2006 accepted 13 November 2006 doi RNA helicases mediate structural rearrangements of RNA or RNA-pro-tein complexes at the expense of ATP hydrolysis. Members of the DEAD box helicase family consist of two flexibly connected helicase domains. They share nine conserved sequence motifs that are involved in nucleotide binding and hydrolysis RNA binding and helicase activity. Most of these motifs line the cleft between the two helicase domains and extensive communication between them is required for RNA unwinding. The two helicase domains of the Bacillus subtilis RNA helicase YxiN were produced separately as intein fusions and a functional RNA helicase was generated by expressed protein ligation. The ligated helicase binds adenine nucleotides with very similar affinities to the wild-type protein. Importantly its intrinsically low ATPase activity is stimulated by RNA and the Michaelis-Menten parameters are similar to those of the wild-type. Finally ligated YxiN unwinds a minimal RNA substrate to an extent comparable to that of the wild-type helicase confirming authentic interdomain communication. In 1990 post-translational excision of intervening sequences from protein precursors was discovered 1 2 . In this self-catalyzed process termed protein splicing the intervening amino-acid sequence intein is excised and the flanking sequences the exteins are .

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