Mutational analysis of the bacteriophage P1 late promoter sequence Ps

Abstract The bacteriophage P1 late promoter sequence P s controls the expression of the genes in the tail-fibre operon. Transcription from P s only occurs during the second half of the P1 vegetative growth cycle and is positively regulated by the product of the phage gene 10. In this study degenerate oligonucleotides were used as primers in site-directed mutagenesis reactions in order to construct a large set of point mutations within the late promoter sequence P s . A total of 35 independent single point mutations was isolated and the mutants were tested for promoter activity. Mutations in the Escherichia coli -like −10 region and in a late operator sequence, containing a symmetric sequence centred around position −22, resulted in significant reductions in promoter strength. Most of these mutations alter base-pairs that are highly conserved among the known late promoter sequences of the P1 family. In addition, insertion mutants that change the spacing between the −10 and the late operator indicate that a special topological arrangement between the two boxes is crucial for late promoter function. These results suggest that the product of gene 10 binds specifically to a late operator in order to activate transcription from P1 late promoter sequences.