Mutations that reduce expression from the P2 promoter of the Escherichia coli galactose operon

Abstract

We describe the isolation and characterisation of twelve different mutations that reduce gene expression from the galP2 promoter, starting with a gal regulatory region with a mutation that inactivated galP1, the cAMP-CRP-dependent promoter. Seven of the new mutations reduce the initiation of transcription at P2 whereas the others reduce translation initiation of the first gal operon gene, galE. Two of the mutations affecting translation fall in the galE initiation codon and the Shine-Dalgarno sequence. Mutations that allow the formation of a stem-loop structure in the messenger including this sequence also reduce translation. A deletion of 11 bp, upstream of the Shine-Dalgarno sequence, almost totally prevents translation. Although none of the point mutations that reduced transcription initiation at P2 fall in the -35 region, we repeatedly isolated insertions in this zone. The point mutations all fell around the -10 region: the strongest effects were found with mutations that altered the sequence away from the consensus that has been established for Escherichia coli promoters. The effects of the two strongest P2 mutations were investigated in the absence of the P1 mutation used for their isolation. One mutation, a T:A to C:G transition at -12, inactivates both P2 and P1. In contrast the other, a T:A to G:C transversion at -19, specifically inactivates P2, but leaves P1 partially active even in the absence of cAMP-CRP. The implications of this are discussed in the context of how cAMP-CRP controls the balance between transcription from P2 and P1 at the gal operon regulatory region.