title: Posttranscriptional activation of the transcriptional activator Rob by dipyridyl in Escherichia coli.
abstract: The transcriptional activator Rob consists of an N-terminal domain (NTD) of 120 amino acids responsible for DNA binding and promoter activation and a C-terminal domain (CTD) of 169 amino acids of unknown function. Although several thousand molecules of Rob are normally present per Escherichia coli cell, they activate promoters of the rob regulon poorly. We report here that in cells treated with either 2,2"- or 4,4"-dipyridyl (the latter is not a metal chelator), Rob-mediated transcription of various rob regulon promoters was increased substantially. A small, growth-phase-dependent effect of dipyridyl on the rob promoter was observed. However, dipyridyl enhanced Rob's activity even when rob was regulated by a heterologous (lac) promoter showing that the action of dipyridyl is mainly posttranscriptional. Mutants lacking from 30 to 166 of the C-terminal amino acids of Rob had basal levels of activity similar to that of wild-type cells, but dipyridyl treatment did not enhance this activity. Thus, the CTD is not an inhibitor of Rob but is required for activation of Rob by dipyridyl. In contrast to its relatively low activity in vivo, Rob binding to cognate DNA and activation of transcription in vitro is similar to that of MarA, which has a homologous NTD but no CTD. In vitro nuclear magnetic resonance studies demonstrated that 2,2"-dipyridyl binds to Rob but not to the CTD-truncated Rob or to MarA, suggesting that the effect of dipyridyl on Rob is direct. Thus, it appears that Rob can be converted from a low activity state to a high-activity state by a CTD-mediated mechanism in vivo or by purification in vitro.
authors.affiliation: Laboratory of Molecular Biology, National Institute of Diabetes, Digestive and Kidney Diseases, National Institutes of Health, Bldg 5. Rm. 333, Bethesda, MD 20892-0560, USA. jlrosner@helix.nih.gov
authors.forename: Judah L
authors.initials: JL
authors.lastname: Rosner
authors.forename: Bindi
authors.initials: B
authors.lastname: Dangi
authors.forename: Angela M
authors.initials: AM
authors.lastname: Gronenborn
authors.forename: Robert G
authors.initials: RG
authors.lastname: Martin
mesh_terms: D015082:2,2'-Dipyridyl / Q000494:pharmacology*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects; D007501:Iron / Q000378:metabolism; D008565:Membrane Proteins; D011401:Promoter Regions, Genetic; D011499:Protein Processing, Post-Translational / Q000187:drug effects*; D011725:Pyridines / Q000494:pharmacology*; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic
chemical_list: D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D011725:Pyridines; C080267:Rob protein, E coli; D015534:Trans-Activators; C062144:inaA protein, Pantoea ananatis; D015082:2,2'-Dipyridyl; D007501:Iron; C034306:4,4'-bipyridyl