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J. Biol. Chem., Vol. 281, Issue 46, 34997-35007, November 17, 2006

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Heterogeneous Nucleotide Occupancy Stimulates Functionality of Phage Shock Protein F, an AAA+ Transcriptional Activator^*

From the Division of Biology, Sir Alexander Fleming Building, Imperial College London, London SW7 2AZ, United Kingdom

The catalytic AAA+ domain (PspF_1–275) of an enhancer-binding protein is necessary and sufficient to contact ⁵⁴-RNA polymerase holoenzyme (E⁵⁴), remodel it, and in so doing catalyze open promoter complex formation. Whether ATP binding and hydrolysis is coordinated between subunits of PspF and the precise nature of the nucleotide(s) bound to the oligomeric forms responsible for substrate remodeling are unknown. We demonstrate that ADP stimulates the intrinsic ATPase activity of PspF_1–275 and propose that this heterogeneous nucleotide occupancy in a PspF_1–275 hexamer is functionally important for specific activity. Binding of ADP and ATP triggers the formation of functional PspF_1–275 hexamers as shown by a gain of specific activity. Furthermore, ATP concentrations congruent with stoichiometric ATP binding to PspF_1–275 inhibit ATP hydrolysis and E⁵⁴-promoter open complex formation. Demonstration of a heterogeneous nucleotide-bound state of a functional PspF_1–275·E⁵⁴ complex provides clear biochemical evidence for heterogeneous nucleotide occupancy in this AAA+ protein. Based on our data, we propose a stochastic nucleotide binding and a coordinated hydrolysis mechanism in PspF_1–275 hexamers.

Received for publication, July 12, 2006 , and in revised form, September 12, 2006.

^* This work was supported n part by project grant funding from the Biotechnology and Biological Sciences Research Council, Swindon, United Kingdom (to M. B.). 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 Recipient of European Molecular Biology Organization Fellowship ALTF 387-2005.

² To whom correspondence should be addressed. Tel.: 44-2075945442; Fax: 44-2075945419; E-mail: m.buck{at}imperial.ac.uk.

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