Cooperative action of the catabolite activator protein and AraC in vitro at the araFGH promoter.
Full activation of transcription of the araFGH promoter, p(FGH), requires both the catabolite activator protein (CAP) and AraC protein. At p(FGH), the binding site for CAP is centered at position -41.5, an essential binding site for AraC is centered at position -79.5, and a second, nonessential binding site is centered at position -154.5. In this work, we used the minimal promoter region required for in vivo activation of p(FGH) to examine the roles of CAP and AraC in stimulating formation of open complexes at p(FGH). Migration retardation assays of open complexes showed that RNA polymerase binds exceptionally tightly to the AraC-CAP-p(FGH) complex and that the order of addition of proteins to the initiating complex is important. Similar assays with RNA polymerase containing truncated alpha subunits suggest that AraC interacts with the C-terminal domain of the alpha subunit. Finally, AraC protein also acts to prevent the improper binding of RNA polymerase at a pseudo promoter near the true p(FGH) promoter.
Johnson
C M
CM
Department of Biology, Johns Hopkins University, Baltimore, Maryland 21218, USA.
Schleif
R F
RF
D000494:Allosteric Regulation / Q000235:genetics; D000495:Allosteric Site; D051777:AraC Transcription Factor; D001426:Bacterial Proteins; D001483:Base Sequence; D001667:Binding, Competitive; D002373:Cyclic AMP Receptor Protein / Q000235:genetics / Q000378:metabolism*; D004247:DNA / Q000235:genetics / Q000378:metabolism; D018983:DNA Footprinting; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics; D005798:Genes, Bacterial / Q000235:genetics*; D007700:Kinetics; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011485:Protein Binding; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism*; D017384:Sequence Deletion / Q000235:genetics; D014157:Transcription Factors; D015533:Transcriptional Activation / Q000235:genetics
D051777:AraC Transcription Factor; C497371:AraC protein, E coli; D001426:Bacterial Proteins; D002373:Cyclic AMP Receptor Protein; D029968:Escherichia coli Proteins; D012097:Repressor Proteins; D014157:Transcription Factors; D004247:DNA; D012321:DNA-Directed RNA Polymerases