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J. Biol. Chem., Vol. 282, Issue 13, 9825-9833, March 30, 2007

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Sensor I Threonine of the AAA+ ATPase Transcriptional Activator PspF Is Involved in Coupling Nucleotide Triphosphate Hydrolysis to the Restructuring of ⁵⁴-RNA Polymerase^*

Jörg Schumacher¹, Nicolas Joly², Mathieu Rappas, Dominic Bradley, Siva R. Wigneshweraraj, Xiaodong Zhang, and Martin Buck³

From the Divisions of Biology and Molecular Biosciences, Imperial College London, London SW7 2AZ, United Kingdom

Transcriptional initiation invariably involves the transition from a closed RNA polymerase (RNAP) promoter complex to a transcriptional competent open complex. Activators of the bacterial⁵⁴-RNAP are AAA+ proteins that couple ATP hydrolysis to restructure the ⁵⁴-RNAP promoter complex. Structures of the ⁵⁴ activator PspF AAA+ domain (PspF_1–275) bound to ⁵⁴ show two loop structures proximal to ⁵⁴ as follows: the ⁵⁴ contacting the GAFTGA loop 1 structure and loop 2 that classifies ⁵⁴ activators as pre-sensor 1 -hairpin AAA+ proteins. We report activities for PspF_1–275 mutated in the AAA+ conserved sensor I threonine/asparagine motif (PspF_1–275^T148A, PspF_1–275^N149A, and PspF_1–275^N149S) within the second region of homology. We show that sensor I asparagine plays a direct role in ATP hydrolysis. However, low hydrolysis rates are sufficient for functional output in vitro. In contrast, PspF_1–275^T148A has severe defects at the distinct step of ⁵⁴ promoter restructuring. This defect is not because of the failure of PspF_1–275^T148A to stably engage with the closed ⁵⁴ promoter, indicating (i) an important role in ATP hydrolysis-associated motions during energy coupling for remodeling and (ii) distinguishing PspF_1–275^T148A from PspF_1–275 variants involved in signaling to the GAFTGA loop 1, which fail to stably engage with the promoter. Activities of loop 2 PspF_1–275 variants are similar to those of PspF_1–275^T148A suggesting a functional signaling link between Thr¹⁴⁸ and loop 2. In PspF_1–275 this link relies on the conserved nucleotide state-dependent interaction between the Walker B residue Glu¹⁰⁸ and Thr¹⁴⁸. We propose that hydrolysis is relayed via Thr¹⁴⁸ to loop 2 creating motions that provide mechanical force to the GAFTGA loop 1 that contacts ⁵⁴.

Received for publication, December 18, 2006 , and in revised form, January 19, 2007.

^* This work was supported in part by the Biotechnology and Biological Sciences Research Council. 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.

² Recipient of EMBO Fellowship ALTF 387-2005.

¹ To whom correspondence may be addressed. Tel.: 44-207-5945366; Fax: 44-207-5842056; E-mail: j.schumacher{at}imperial.ac.uk. ³ To whom correspondence may be addressed. Tel.: 44-207-5945422; Fax: 44-207-5945419; E-mail: m.buck{at}imperial.ac.uk.

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