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Nucleic Acids Research, 1988, Vol. 16, No. 15 7315-7332
© 1988

Articles

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

T. Morita, K. Shigesada1, F. Kimizuka2 and H. Aiba*,

Department of Chemistry, University of Tsukuba Tsukuba 305, Japan 1Institute for Virus Research, Kyoto University Kyoto 606, Japan 2Central Research Laboratory Takara Shuzo Co., Ltd., Otsu 520-01, Japan

*To whom correspondence should be addressed

Received May 24, 1988. Accepted June 27, 1988.

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 ß-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.


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