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J. Biol. Chem., Vol. 283, Issue 9, 5567-5576, February 29, 2008

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Allosteric Activation of the ATPase Activity of the Escherichia coli RhlB RNA Helicase^*

Jonathan A. R. Worrall¹, Françoise S. Howe, Adam R. McKay², Carol V. Robinson, and Ben F. Luisi³

From the Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge CB2 1GA and the Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, United Kingdom

Helicase B (RhlB) is one of the five DEAD box RNA-dependent ATPases found in Escherichia coli. Unique among these enzymes, RhlB requires an interaction with the partner protein RNase E for appreciable ATPase and RNA unwinding activities. To explore the basis for this activating effect, we have generated a di-cistronic vector that overexpresses a complex comprising RhlB and its recognition site within RNase E, corresponding to residues 696–762. Complex formation has been characterized by isothermal titration calorimetry, revealing an avid, enthalpy-favored interaction between the helicase and RNase E-(696–762) with an equilibrium binding constant (K_a) of at least 1 x 10⁸ M^-1. We studied ATPase activity of mutants with substitutions within the ATP binding pocket of RhlB and on the putative interaction surface that mediates recognition of RNase E. For comparisons, corresponding mutations were prepared in two other E. coli DEAD box ATPases, RhlE and SrmB. Strikingly, substitutions at a phenylalanine near the Q-motif found in DEAD box proteins boosts the ATPase activity of RhlB in the absence of RNA, but completely inhibits it in its presence. The data support the proposal that the protein-protein and RNA-binding surfaces both communicate allosterically with the ATPase catalytic center. We conjecture that this communication may govern the mechanical power and efficiency of the helicases, and is tuned in individual helicases in accordance with cellular function.

Received for publication, October 17, 2007 , and in revised form, December 21, 2007.

^* This work was supported by the Wellcome Trust. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Current address: Dept. of Biological Sciences, University of Essex, Wivenhoe Park, Colchester CO4 3SQ, UK.

² Current address: Dept. of Chemistry, University College London, 20 Gordon St., London WC1H 0AJ, UK.

³ To whom correspondence should be addressed. Tel.: 44-1223-766019; E-mail: bfl20{at}mole.bio.cam.ac.uk.

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