A conserved region in the sigma54-dependent activator DctD is involved in both binding to RNA polymerase and coupling ATP hydrolysis to activation.
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.
Wang
Y K
YK
Department of Microbiology, University of Georgia, Athens 30602, USA.
Lee
J H
JH
Brewer
J M
JM
Hoover
T R
TR
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
D001426:Bacterial Proteins; D014157:Transcription Factors; C073788:dctD protein, Rhizobium meliloti; C071989:APT; D012321:DNA-Directed RNA Polymerases; D017259:Dimaprit