Alterations within the activation domain of the sigma 54-dependent activator DctD that prevent transcriptional activation.
Rhizobium meliloti DctD (C4-dicarboxylate transport protein D) is a transcriptional activator that catalyzes the ATP-dependent isomerization of closed complexes between sigma 54-RNA polymerase holoenzyme and the dctA promoter to open complexes. Following random mutagenesis of dctD, 55 independent mutant forms of DctD that failed to activate transcription from a dctA'-'lacZ reporter gene in Escherichia coli were selected, and the amino acid substitutions were determined for these mutant proteins. Amino acid substitutions were distributed throughout the central domain of the protein, the domain responsible for transcription activation, but most of the substitutions occurred within three highly conserved regions of the protein. Selected mutant proteins were purified, and their activities were studied in vitro. All of the purified mutant proteins appeared to have normal DNA-binding activity and interacted with sigma 54 and core RNA polymerase, as determined from protein crosslinking assays. Proteins with amino acid substitutions in a region spanning amino acid positions 222 to 225 retained their ATPase activities, whereas proteins with substitutions in other regions had little or no ATPase activity. Taken together, these data suggest that the region that encompasses amino acid residues 222 through 225 probably functions in coupling the energy released from ATP hydrolysis to open complex formation rather than as a major determinant for binding to RNA polymerase.
Wang
Y K
YK
Department of Microbiology, University of Georgia, Athens 30602, USA.
Hoover
T R
TR
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D001665:Binding Sites; D018983:DNA Footprinting; D004268:DNA-Binding Proteins / Q000235:genetics; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D011485:Protein Binding; D017384:Sequence Deletion; D012808:Sigma Factor / Q000235:genetics*; D016962:Sinorhizobium meliloti / Q000235:genetics*; D013329:Structure-Activity Relationship; D014157:Transcription Factors / Q000235:genetics*; D014158:Transcription, Genetic
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D046911:Macromolecular Substances; D012808:Sigma Factor; D014157:Transcription Factors; C073788:dctD protein, Rhizobium meliloti; D000255:Adenosine Triphosphate; D012321:DNA-Directed RNA Polymerases