Characterization of the AAA+ domain of Sinorhizobium meliloti DctD, a sigma 54-dependent transcriptional activator
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Transcription initiation by s54-RNA polymerase holoenzyme (s54-holoenzyme) requires an activator that couples ATP hydrolysis to formation of an open promoter complex. These activators are modular, consisting of an N-terminal regulatory domain, a C-terminal DNAbinding 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) was sufficient to activate transcription in vivo and in vitro. Many of the transcripts produced in vitro terminate prematurely suggesting that the DctD AAA+ domain interfered with transcription elongation. Overexpression of the DctD AAA+ domain in Escherichia coli inhibited transcriptional activation, which may have resulted from interference with transcriptional elongation or titration of s54. The DctD AAA+ domain migrated as a single, highmolecular weight species in a native gel and formed a stable complex with s54 in the presence of ADP-aluminum fluoride. In contrast, a DctD protein consisting of the AAA+ and DNA-binding domains displayed several oligomeric species in the native gel and failed to bind s54. Mutational analysis of the AAA+ domain of DctD produced mutants in two conserved regions that activated transcription efficiently from a reporter gene that had an upstream activation sequence (UAS), but not from a reporter gene that lacked the UAS. Activities of these UAS-dependent mutants were stimulated when the UAS was provided in trans, suggesting that the UAS acted as a positive allosteric effector. Taken together, these data suggest that the DNA-binding domain of DctD inhibits formation of functional oligomers of the AAA+ domain and modulates interactions of this domain with s54. Finally, several substitutions in a potential hinge region between the a/ß and a-helical subdomains of the DctD AAA+ domain resulted in mutant proteins that were defective in coupling ATP hydrolysis to transcriptional activation. The a- helical domain contains the sensor II helix that is involved in nucleotide sensing in other AAA+ proteins. Substitutions in this hinge region may relocate the sensor II helix so that it cannot sense the nucleotide or they may interfere with movement of the sensor II helix in response to ATP hydrolysis.