Deletion analysis of the CAP-cAMP binding site of the Escherichia coil lactose promoter

• Published in: Nucleic acids research 1984-07, Vol.12 (13), p.5449-5464
• Main Authors: Yu, Xian-Ming, Reznikoff, William S.
• Format: Article
• Language: English
• ISSN: 0305-1048; 1362-4962
• DOI: 10.1093/nar/12.13.5449

S1 nuclease was used to generate a series of deletions which extend into tAe CAP-cANP binding site from upstream of the Escherichia coli lactose operon promoter (lacP). Deletion and insertion mutations were also created which changed the spacing region between the CAP-cAMP binding site and the lacP -35 region. The promoter activities of these mutations were compared by measuring the levels of β-galactosidase gene expression in vivo. The results show that sequence information prior to 74 base pairs (−74) upstream from the transcription start site (designated as +1) is not necessary for the full activation of the lac promoter by the CAP-cAMP complex. However, the deletion which extends to the −71 position retains only one third of the promoter activity in the presence of the CAP-cAMP complex. Removal of one symmetrical element from the two fold symmetry in the CAP-cAMP binding site abolished the CAP-cAMP stimulation of the lac promoter. Spacer muta tions which increase by one base pair or decrease by two base pairs the length of the spacing region between the CAP-cAMP binding site and the lacP-35 region drastically reduced the CAP-cAMP stimulation of the lac promoter. This suggests that the distance between the lac promoter transcription start site and CAP-cAMP binding site is crucial for the function of the lac promoter, despite the fact that this distance varies in other E. coli promoters positively regulated by CAP-cAMP. A deletion which extends to the −59 position results in a two fold enhanced expression of lac in the absence of CAP-cAMP. This is consistent with the existance of a competitive RNA polymerase binding site in this region which would normally act to inhibit RNA polymerase binding.