title: Heterologous cooperativity in Escherichia coli. The CytR repressor both contacts DNA and the cAMP receptor protein when binding to the deoP2 promoter.
abstract: Promoters in Escherichia coli that are negatively regulated by the CytR repressor are also activated by the cAMP receptor protein (CRP) complexed to cAMP; as a characteristic, these promoters encode two binding sites for the cAMP.CRP complex. Biochemical and genetic studies have shown that CytR relies on interactions with the cAMP.CRP complex in order to bind promoter DNA and repress transcription. Here we have purified CytR to near homogeneity and addressed the question of how it interacts with the deoP2 promoter. Gel retardation and DNase I footprinting analyses show that CytR is a true sequence-specific DNA-binding protein that binds to the sequence between the two CRP sites in deoP2 with a relatively low affinity. In the presence of the cAMP.CRP complex the two protein species bind cooperatively to deoP2, forming a complex in which CytR occupies the sequence between the two DNA bound cAMP.CRP complexes. Furthermore, the inducer (cytidine) does not affect independent DNA binding of CytR, rather the CytR/cAMP.CRP cooperativity is perturbed. These results indicate that CytR binding to deoP2 relies on both repressor-DNA interactions and protein-protein interactions to cAMP.CRP. This combinatorial repression mechanism, in which an activator functions as an adaptor for a repressor that is not capable of blocking transcription on its own, is unprecedented in prokaryotes; it is, however, reminiscent of repression mechanisms found in eukaryotes.
authors.affiliation: Department of Molecular Biology, Odense University, Denmark.
authors.forename: H
authors.initials: H
authors.lastname: Pedersen
authors.forename: L
authors.initials: L
authors.lastname: Søgaard-Andersen
authors.forename: B
authors.initials: B
authors.lastname: Holst
authors.forename: P
authors.initials: P
authors.lastname: Valentin-Hansen
mesh_terms: D001426:Bacterial Proteins / Q000378:metabolism*; D001483:Base Sequence; D002373:Cyclic AMP Receptor Protein / Q000302:isolation & purification / Q000378:metabolism*; D016172:DNA Fingerprinting; D004269:DNA, Bacterial / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D012097:Repressor Proteins / Q000378:metabolism*; D014158:Transcription, Genetic
chemical_list: D001426:Bacterial Proteins; D002373:Cyclic AMP Receptor Protein; C048425:CytR protein, E coli; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D012097:Repressor Proteins