Purification and characterization of the AAA+ domain of Sinorhizobium meliloti DctD, a σ54-dependent transcriptional activator

Hao Xu, Baohua Gu, B. Tracy Nixon, Timothy R. Hoover

Research output: Contribution to journalArticle

22 Scopus citations

Abstract

Activators of σ54-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(Δ1-142) a truncated DctD protein consisting of the AAA+ and DNA-binding domains. In addition, the DctD AAA+ domain was more homogeneous than DctD(Δ1-142) when analyzed by native gel electrophoresis, migrating predominantly as a single high-molecular-weight species, while DctD(Δ1-142) displayed multiple species. The DctD AAA+ domain, but not DctD(Δ1-142), formed a stable complex with σ 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 σ54 in addition to its role in recognition of upstream activation sequences.

Original languageEnglish (US)
Pages (from-to)3499-3507
Number of pages9
JournalJournal of bacteriology
Volume186
Issue number11
DOIs
StatePublished - Jun 2004

All Science Journal Classification (ASJC) codes

  • Microbiology
  • Molecular Biology

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