title: Single amino acid substitutions in the cAMP receptor protein specifically abolish regulation by the CytR repressor in Escherichia coli.
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 tandem binding sites for cAMP-CRP. In one such promoter, deoP2, CytR binds to the region between the tandem CRP binding sites with a relatively low affinity; in the presence of cAMP-CRP, however, the repressor and activator bind cooperatively to the DNA. Here we have investigated this cooperativity by isolating mutants of the CRP protein that abolish CytR regulation without exhibiting a concomitant loss in their ability to activate transcription. Four different, single amino acid substitutions in CRP give rise to this phenotype. These amino acids lie in close proximity on the surface of the CRP tertiary structure in a portion of the protein that is not in contact with the DNA. In vitro analyses of one of the CRP mutants show that it interacts with the DNA in a manner indistinguishable from wild-type CRP, whereas its interaction with CytR is perturbed. These results strongly indicate that cooperative DNA binding of CytR and cAMP-CRP is achieved through protein-protein interactions.
authors.affiliation: Department of Molecular Biology, Odense University, Denmark.
authors.forename: L
authors.initials: L
authors.lastname: Søgaard-Andersen
authors.forename: A S
authors.initials: AS
authors.lastname: Mironov
authors.forename: H
authors.initials: H
authors.lastname: Pedersen
authors.forename: V V
authors.initials: VV
authors.lastname: Sukhodelets
authors.forename: P
authors.initials: P
authors.lastname: Valentin-Hansen
mesh_terms: D001426:Bacterial Proteins / Q000378:metabolism*; D001483:Base Sequence; D003001:Cloning, Molecular; D002373:Cyclic AMP Receptor Protein / Q000235:genetics / Q000378:metabolism*; D003850:Deoxyribonuclease I; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins; D046911:Macromolecular Substances; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000378:metabolism; D012097:Repressor Proteins / Q000378:metabolism*; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D002373:Cyclic AMP Receptor Protein; C048425:CytR protein, E coli; D029968:Escherichia coli Proteins; D046911:Macromolecular Substances; D011994:Recombinant Proteins; D012097:Repressor Proteins; D003850:Deoxyribonuclease I; D001616:beta-Galactosidase