Purification and characterization of the AAA+ domain of Sinorhizobium meliloti DctD, a sigma54-dependent transcriptional activator.
Activators of sigma54-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(Delta(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(Delta(1-142)) when analyzed by native gel electrophoresis, migrating predominantly as a single high-molecular-weight species, while DctD(Delta(1-142)) displayed multiple species. The DctD AAA+ domain, but not DctD(Delta(1-142)), formed a stable complex with sigma54 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 sigma54 in addition to its role in recognition of upstream activation sequences.
Xu
Hao
H
Department of Microbiology, University of Georgia, Athens, Georgia 30602, USA.
Gu
Baohua
B
Nixon
B Tracy
BT
Hoover
Timothy R
TR
D001426:Bacterial Proteins / Q000737:chemistry / Q000302:isolation & purification*; D001665:Binding Sites; D004268:DNA-Binding Proteins; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry / Q000502:physiology*; D017433:Protein Structure, Secondary; D051877:RNA Polymerase Sigma 54; D012808:Sigma Factor / Q000737:chemistry / Q000502:physiology*; D016962:Sinorhizobium meliloti / Q000737:chemistry*; D015534:Trans-Activators / Q000302:isolation & purification*; D014157:Transcription Factors / Q000737:chemistry / Q000302:isolation & purification*
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D012808:Sigma Factor; D015534:Trans-Activators; D014157:Transcription Factors; C073788:dctD protein, Rhizobium meliloti; D012321:DNA-Directed RNA Polymerases; D051877:RNA Polymerase Sigma 54