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.
Highlights
We have used an S1 mapping assay to demonstrate utilize galactose [6].in vitro transcription with the that the mRNA transcripts of the Escherichia coli ga- DNA of this mutant indicated an inactive CAMP-CRP-delactose operon found in intaEct. coli cells with a defect pendent promoter, P I, butan active Pzpromoter
The Pz in adenylate cyclasoer the cyclicAMP receptor protein promoter was inhibited by CAMP-CRP ( 6 ) .with this contain at thei5r' end about five nucleotides more thamnutant, both in vitro and in vivo, more gal expression was the gal mRNA molecules made in wild type cells
Gal cistron, were equal or higher in cya- cells than in cya' cells, whereas the levels of galactokinase,the enzyme encoded by the promoter distal cistron, are much lower in cya- than in cya' cells [8].3These enzyme measurements were interpreted by Ullmann et al [8,9]as indicating that thesynthesis of gal epimerase was independent of CAMP-CRPand that only one gal promoter was functional in vivo
Summary
We have used an S1 mapping assay to demonstrate utilize galactose [6].in vitro transcription with the that the mRNA transcripts of the Escherichia coli ga- DNA of this mutant indicated an inactive CAMP-CRP-delactose operon found in intaEct. coli cells with a defect pendent promoter, P I , butan active Pzpromoter. (i) In vitro transcription experiments with DNA fragments containing the operator-promoter sequences showed the existence of termination mechanism, and implied that our previous in vitro transcription results, which indicated the existence of two distinct gal promoters, might not correspond to the situation found in intact cells. This interpretation does not take into account the results with the gal promoter mutant ( 6 )discussed in the preceding paragraph.
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