Regulatory effect of a synthetic CRP recognition sequence placed downstream of a promoter

A series of plasmids were constructed in which a promoter was introduced into a lac-based operon fusion vector. A perfectly symmetrical oligonucleotide of 22-bp corresponding to an idealized binding site for cAMP receptor protein (CRP) of E. coli was chemically synthesized. The synthetic CRP site was placed between the promoter and the lacZ structural gene with varying distances from the promoter. Specific binding of cAMP-CRP complex to the synthetic CRP site was shown by a gel retardation and a DNase I footprinting assays. Plasmid constructs were transformed into crp+ and crp- cells carrying a chromosomal deletion of the lac genes. The regulatory effect of the inserted CRP site was examined by comparing the beta-galactosidase activity and the levels of RNA transcript in two cells harboring the plasmids. We found a strong inhibitory effect of the CRP site in the presence of cAMP and CRP when it was placed close to the promoter. When the CRP site was placed far downstream of the promoter, a moderate repression of transcription was observed.