The selection and characterisation of two novel mutations in the overlapping promoters of the Escherichia coli galactose operon.

Mutations that result in small decreases or increases in expression from the Escherichia coli galactose operon promoter region can be detected by using a plasmid in which the gal promoters were fused to the lac operon. We describe how the level of lac expression was adjusted so that the Lac phenotype of host cells was optimally sensitive to changes in the gal promoter sequence. We have investigated the properties of two new gal promoter mutations both in vivo and in vitro, and have determined their effects on the two overlapping gal promoters, P1 and P2. Although one mutation causes only a small reduction in overall expression in vivo, it completely suppresses transcription initiation at the P1 promoter. However, it also increases expression from the P2 promoter, which compensates for the change at P1. This mutation, a GC to AT transition, falls in a zone just upstream of the P1 Pribnow box, which is essential for P1 activity, whilst improving the homology between the P2 Pribnow box and the consensus sequence. The second mutation causes a small increase in P1 activity. This change, a GC to AT transition at -23, falls in the spacer region between the Pribnow box and the -35 region, a zone containing no known promoter consensus sequences. We suggest that this mutation, which creates a stretch of five AT base pairs, acts by increasing the twist angle of the sequences in the spacer region. We argue that the increase in promoter activity is due to this twist changing the relative orientation of the Pribnow box and -35 regions.