The bacteriophage T4 transcriptional activator MotA accepts various base-pair changes within its binding sequence

During infection, bacteriophage T4 regulates three sets of genes: early, middle, and late. The host RNA polymerase is capable of transcribing early genes, but middle transcription requires the T4-encoded transcriptional activator, MotA protein, and the T4 co-activator, AsiA protein, both of which bind to the sigma 70 (sigma70) subunit of RNA polymerase. MotA also binds a DNA sequence (a MotA box), centered at position -30. The identification of more than 20 middle promoters suggested that a strong match to the MotA box consensus sequence (t/a)(t/a)TGCTT(t/c)A was critical for MotA activation. We have investigated how specific base changes within the MotA box sequence affect MotA binding and activation in vitro, and we have identified seven new middle promoters in vivo. We find that an excellent match to the sigma70 -10 consensus sequence, rather than an excellent match to the MotA box consensus sequence, is an invariant feature of MotA-dependent promoters. Many single base changes in the MotA box are tolerated in binding and activation assays, indicating that there is more flexibility in the sequence requirements for MotA than was previously appreciated. We also find that using the natural T4 DNA, which contains glucosylated, 5-hydoxymethylated cytosine residues, affects the ability of particular MotA box sequences to activate transcription. We suggest that MotA and AsiA may function like certain eukaryotic TAFs (TATA binding protein (TBP) associated factors) whose binding to TBP results in transcription from new core promoter sequences.