Deletion mutagenesis of the Escherichia coli galactose operon promoter region

Abstract

Using recombinant DNA technology we have created a series of progressively longer deletions both upstream and downstream from the Escherichia coli galactose operon regulatory region. The effects of these lesions on expression of the two overlapping galactose promoters have been quantitated after DNA fragments carrying these deletions were cloned in a plasmid vector, in which the beta-galactosidase gene could be expressed from the truncated galactose regulatory region. The results allow us to determine which sequences are necessary for the activity of the two promoters. Our results show that for the P1 promoter, which is controlled by the cyclic AMP-cyclic AMP receptor protein complex (cAMP-CRP), the sequence necessary for full activity starts 56 base-pairs upstream from the transcription initiation point. In contrast, for the P2 promoter, which functions in the absence of cAMP-CRP, the crucial sequence extends to only 39 base-pairs upstream from the transcription start. Deletions that cut into these sequences cause reductions in promoter strength, although some promoter activity is observed even when the "-35 region" of both P2 and P1 are deleted. Analysis of deletions originating downstream from the regulatory region shows that the elimination of the P1 and P2 Pribnow box sequences leads to loss of promoter activity. However, sequences downstream from the P1 start can be replaced without affecting the activity of either promoter. Finally examination of DNA fragments containing total deletions of both galactose promoters allows us to confirm that the flanking sequences contain no significant promoter activity and that the P1 and P2 promoters are principally responsible for galactose operon expression in vivo.