title: A conserved region in the sigma54-dependent activator DctD is involved in both binding to RNA polymerase and coupling ATP hydrolysis to activation.
abstract: Rhizobium melioti DctD activates transcription from the dctA promoter by catalysing the isomerization of closed complexes between sigma54-RNA polymerase holoenzyme and the promoter to open complexes. DctD must make productive contact with sigma54-holoenzyme and hydrolyse ATP to catalyse this isomerization. To define further the activation process, we sought to isolate mutants of DctD that had reduced affinities for sigma54-holoenzyme. Mutagenesis was confined to the well-conserved C3 region of the protein, which is required for coupling ATP hydrolysis to open complex formation in sigma54-dependent activators. Mutant forms of DctD that failed to activate transcription and had substitutions in the C-terminal half of the C3 region were efficiently cross-linked to sigma54 and the beta-subunit of RNA polymerase, suggesting that they bound normally to sigma54-holoenzyme. In contrast, some mutant forms of DctD with amino acid substitutions in the N-terminal half of the C3 region had reduced affinities for sigma54 and the beta-subunit in the cross-linking assay. These data suggest that the N-terminal half of the C3 region of DctD contains a site that may contact sigma54-holoenzyme during open complex formation.
authors.affiliation: Department of Microbiology, University of Georgia, Athens 30602, USA.
authors.forename: Y K
authors.initials: YK
authors.lastname: Wang
authors.forename: J H
authors.initials: JH
authors.lastname: Lee
authors.forename: J M
authors.initials: JM
authors.lastname: Brewer
authors.forename: T R
authors.initials: TR
authors.lastname: Hoover
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D017124:Conserved Sequence; D012321:DNA-Directed RNA Polymerases / Q000235:genetics / Q000378:metabolism*; D017259:Dimaprit / Q000031:analogs & derivatives* / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D006868:Hydrolysis; D008969:Molecular Sequence Data; D009154:Mutation; D016415:Sequence Alignment; D016962:Sinorhizobium meliloti / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D014157:Transcription Factors; C073788:dctD protein, Rhizobium meliloti; C071989:APT; D012321:DNA-Directed RNA Polymerases; D017259:Dimaprit