Purification and Characterization of the AAA+ Domain ofSinorhizobium melilotiDctD, a σ54-Dependent Transcriptional Activator
Open Access
- 1 June 2004
- journal article
- Published by American Society for Microbiology in Journal of Bacteriology
- Vol. 186 (11) , 3499-3507
- https://doi.org/10.1128/jb.186.11.3499-3507.2004
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 ofSinorhizobium melilotiC4-dicarboxylic acid transport protein D (DctD) is sufficient to activate transcription. Deletion analysis of the 3′ end ofdctDidentified 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 σ54in 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 σ54in addition to its role in recognition of upstream activation sequences.Keywords
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