A role for the interdomain linker region of the Escherichia coli CytR regulator in repression complex formation.

Regulatory complexes formed by the CytR repressor protein and the cAMP receptor protein (CRP) prevent transcription initiation from several promoters in Escherichia coli. The formation of the complexes is mediated by protein-DNA interactions and protein-protein interactions between the two regulators. Interestingly, co-binding with CRP has a profound effect on the configuration of the DNA-binding targets preferred by CytR. When binding to DNA by itself, CytR binds preferentially to two octamer repeats in direct or inverted orientation, and separated by 2 bp. However, in the presence of CRP, CytR recognizes inverted repeats separated by 10-13 bp, or direct repeats separated by 1 bp. A fixed orientation of at least one CytR octamer repeat in close proximity to a CRP-binding target is a common architectural feature at promoters optimised for repression complex formation. These observations suggest that CRP alters the DNA-binding mode of CytR. Here, we have investigated the CRP-induced changes in CytR by protein footprinting and alanine-scanning mutagenesis. Our data suggest that a flexible interdomain linker region in CytR, connecting the DNA-binding domain to the dimerization domain allows the repressor protein to interact with DNA-binding sites in a highly relaxed manner, as shown previously, and plays an active role in transcription regulation. Thus, the interactions between CRP, CytR and DNA within the repression complex appear to be more extensive than anticipated. The results support and extend the view that the high degree of adaptability observed in the CytR/CRP regulatory system is obtained though multiple adjustable interactions between the implicated factors.