Abstract

We have used an S1 mapping assay to demonstrate that the mRNA transcripts of the Escherichia coli galactose operon found in intact E. coli cells with a defect in adenylate cyclase or the cyclic AMP receptor protein contain at their 5' end about five nucleotides more than the gal mRNA molecules made in wild type cells.The same difference between gal RNA synthesized in vitro in the absence of cyclic AMP or cyclic AMP receptor protein and gal RNA made in the presence of these factors is detected by this assay.Our results strongly suggest that the same two overlapping promoters, which we previously identified by in vitro transcription of gal DNA fragments, also control the expression of the galactose operon in intact cells.The intracellular levels of cyclic AMP determine which promoter is utilized.The regulatory elements of the promoter-operator segment of the galactose operon of Escherichia coli have a unique arrangement.First, the operator is located about 60 bases preceding the start site for the CAMP-dependent transcript in contrast to the location of the operator in other bacterial or bacteriophage operons (1).Second, the cyclic AMP receptor protein binds in the -35 region in gal (2), whereas in lac it binds between -50 and -74 (3), and in ara around -85 (4).In gal, the sequences upstream of -59 can, in fact, be replaced by unrelated DNA sequences without changing the CAMP-CRP'-dependent regulation of gal.' Third, a number of observations led us to propose that two promoters, which overlap each other, control the expression of the operon (6).(i) In vitro transcription experiments with DNA fragments containing the operator-promoter sequences showed the existence of two start sites.Transcription from one site (SI) is dependent on CAMP-CRP, whereas transcription from the second site ( S Z ) occurs in the absence of CAMP-CRP.The two start sites are separated by five base pairs or half a turn in the DNA helix, and are each preceded by Pribnow box sequences.(ii) A gal promoter mutant, which maps at -11 in the Pribnow box corresponding to the CAMP-CRP-dependent start site, is unable to utilize galactose in cya+ cells.Introduction of a cyamutation restores, however, the ability of this gal mutant to