title: Design of CytR regulated, cAMP-CRP dependent class II promoters in Escherichia coli: RNA polymerase-promoter interactions modulate the efficiency of CytR repression.
abstract: In CytR regulated promoters in Escherichia coli, the cAMP-CRP complex acts as a transcriptional activator as well as a co-repressor for the CytR protein. Repression by CytR depends on the formation of nucleoprotein complexes in which CytR binds cooperatively to the DNA with one or two cAMP-CRP complexes. Here, we demonstrate that in order to establish CytR regulation in a cAMP-CRP dependent class II promoter with a single CRP site (CRP site centred around position -40.5) in which the CytR operator is located upstream of the CRP site, high affinity binding sites for both regulators are required. The efficiency of CytR regulation was observed to be modulated by RNA polymerase (RNAP)-promoter interactions. Specifically, in class II promoters with a single CRP site, the efficiency of CytR regulation was found to correlate inversely with cAMP-CRP independent promoter activity. These observations can be reconciled in a competition model for CytR regulation in which CytR and RNAP compete for cooperative binding with cAMP-CRP to the promoters in vivo. In this model, two mutually exclusive ternary complexes can be formed: a CytR/cAMP-CRP/promoter repression complex and an RNAP/cAMP-CRP/promoter activation complex. Thus, CytR regulation critically depends on formation of a repression complex that binds the promoter with sufficiently high affinity to exclude formation of the competing activation complex. We suggest that the transition from repression to activation involves a switch in the protein-protein interactions made by cAMP-CRP from CytR to RNAP. On the basis of the regulatory features of the promoters analysed here, we speculate about the advantages offered by the structural complexity of natural CytR/cAMP-CRP regulated promoters.
authors.affiliation: Department of Molecular Biology, Odense University, Denmark.
authors.forename: H H
authors.initials: HH
authors.lastname: Kristensen
authors.forename: P
authors.initials: P
authors.lastname: Valentin-Hansen
authors.forename: L
authors.initials: L
authors.lastname: Søgaard-Andersen
mesh_terms: D001483:Base Sequence; D001665:Binding Sites; D002352:Carrier Proteins; D002373:Cyclic AMP Receptor Protein / Q000378:metabolism*; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins; D015964:Gene Expression Regulation, Bacterial; D008957:Models, Genetic; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009876:Operon; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011485:Protein Binding; D012097:Repressor Proteins / Q000378:metabolism*
chemical_list: D002352:Carrier Proteins; D002373:Cyclic AMP Receptor Protein; C048425:CytR protein, E coli; D029968:Escherichia coli Proteins; D012097:Repressor Proteins; D012321:DNA-Directed RNA Polymerases