Middle promoters constitute the most abundant and diverse class of promoters in bacteriophage T4

The temporally regulated transcription program of bacteriophage T4 relies upon the sequential utilization of three classes of promoters: early, middle and late. Here we show that middle promoters constitute perhaps the largest and the most diverse class of T4 promoters. In addition to 45 T4 middle promoters known to date, we mapped 13 new promoters, 10 of which deviate from the consensus MotA box, with some of them having no obvious match to it. So, 30 promoters of 58 identified now deviate from the consensus sequence deduced previously. In spite of the differences in their sequences, the in vivo activities of these T4 middle promoters were demonstrated to be dependent on both activators, MotA and AsiA. Traditionally, the MotA box was restricted to a 9 bp sequence with the highly conserved motif TGCTT. New logo based on the sequences of currently known middle promoters supports the conclusion that the consensus MotA box is comprised of 10 bp with the highly conserved central motif GCT. However, some apparently good matches to the consensus of middle promoters do not produce transcripts either in vivo or in vitro, indicating that the consensus sequence alone does not fully define a middle promoter.