DESIGN OF CYTR REGULATED, CAMP-CRP DEPENDENT CLASS-II PROMOTERS IN ESCHERICHIA-COLI - RNA POLYMERASE-PROMOTER INTERACTIONS MODULATE THE EFFICIENCY OF CYTR REPRESSION

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 nucleopro tein complexes in which CytR binds cooperatively to the DNA with one o r two cAMP-CRP complexes. Here, we demonstrate that in order to establ ish CytR regulation in a cAMP-CRP dependent class II promoter with a s ingle CRP site (CRP site centred around position -40.5) in which the C ytR operator is located upstream of the CRP site, high affinity bindin g sites for both regulators are required. The efficiency of CytR regul ation was observed to be modulated by RNA polymerase (RNAP)-promoter i nteractions. Specifically, in class II promoters with a single CRP sit e, 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 a nd 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/cAM P-CRP/promoter activation complex. Thus, CytR regulation critically de pends on formation of a repression complex that binds the promoter wit h sufficiently high affinity to exclude formation of the competing act ivation complex. We suggest that the transition from repression to act ivation 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 offere d by the structural complexity of natural CytR/cAMP-CRP regulated prom oters. (C) 1997 Academic Press Limited.