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Journal of Bacteriology, June 2004, p. 3499-3507, Vol. 186, No. 11
0021-9193/04/$08.00+0     DOI: 10.1128/JB.186.11.3499-3507.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Purification and Characterization of the AAA+ Domain of Sinorhizobium meliloti DctD, a ⁵⁴-Dependent Transcriptional Activator

Hao Xu,^1, Baohua Gu,^2, B. Tracy Nixon,² and Timothy R. Hoover^1*

Department of Microbiology, University of Georgia, Athens, Georgia,¹ Department of Biochemistry and Molecular Biology, Pennsylvania State University, University Park, Pennsylvania²

Received 10 October 2003/ Accepted 18 February 2004

Activators of ⁵⁴-RNA polymerase holoenzyme couple ATP hydrolysis to formation of an open complex between the promoter and RNA polymerase. These activators are modular, consisting of an N-terminal regulatory domain, a C-terminal DNA-binding domain, and a central activation domain belonging to the AAA+ superfamily of ATPases. The AAA+ domain of Sinorhizobium meliloti C₄-dicarboxylic acid transport protein D (DctD) is sufficient to activate transcription. Deletion analysis of the 3' end of dctD identified the minimal functional C-terminal boundary of the AAA+ domain of DctD as being located between Gly-381 and Ala-384. Histidine-tagged versions of the DctD AAA+ domain were purified and characterized. The DctD AAA+ domain was significantly more soluble than DctD_(1-142), a truncated DctD protein consisting of the AAA+ and DNA-binding domains. In addition, the DctD AAA+ domain was more homogeneous than DctD_(1-142) when analyzed by native gel electrophoresis, migrating predominantly as a single high-molecular-weight species, while DctD_(1-142) displayed multiple species. The DctD AAA+ domain, but not DctD_(1-142), formed a stable complex with ⁵⁴ in the presence of the ATP transition state analogue ADP-aluminum fluoride. The DctD AAA+ domain activated transcription in vitro, but many of the transcripts appeared to terminate prematurely, suggesting that the DctD AAA+ domain interfered with transcription elongation. Thus, the DNA-binding domain of DctD appears to have roles in controlling the oligomerization of the AAA+ domain and modulating interactions with ⁵⁴ in addition to its role in recognition of upstream activation sequences.

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* Corresponding author. Mailing address: 527 Biological Sciences Building, University of Georgia, Athens, GA 30602. Phone: (706) 542-2675. Fax: (706) 542-2674. E-mail: trhoover{at}uga.edu.

Present address: Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA 30602.

Present address: Department of Virology, The Metabolic and Viral Diseases Center of Excellence for Drug Discovery, GlaxoSmithKline Pharmaceuticals, Collegeville, Pa.

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Journal of Bacteriology, June 2004, p. 3499-3507, Vol. 186, No. 11
0021-9193/04/$08.00+0     DOI: 10.1128/JB.186.11.3499-3507.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

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