Binding of the bacteriophage T4 transcriptional activator, MotA, to T4 middle promoter DNA: evidence for both major and minor groove contacts.

During infection, the bacteriophage T4 transcriptional activator MotA, the co-activator AsiA, and host RNA polymerase are needed to transcribe from T4 middle promoters. Middle promoters contain a -10 region recognized by the sigma(70)subunit of RNA polymerase and a MotA box centered at -30 that is bound by MotA. We have investigated how the loss or modification of base determinants within the MotA box sequence 5'TTTGCTTTA3' (positions -34 to -26 of a middle promoter) affects MotA function. Gel retardation assays with mutant MotA boxes are consistent with the idea that MotA uses minor groove contacts upstream and major groove contacts downstream of the center GC, and does not require any specific base feature at the C.G base-pair at position -30. In particular, the 5-methyl residue on the thymine residue at position -29, a major groove contact, contributes to MotA binding, while converting the T.A at -32 to a C. I base-pair, a change that affects the major but nor the minor groove, yields a MotA box that is similar to wild-type. However, methylation interference analyses indicate that neither the binding of MotA nor the binding of polymerase/MotA/AsiA to the middle promoter PuvsXis inhibited by premethylation of guanine and adenine residues, suggesting that binding does not require minor groove contact with any specific T.A base-pair. Using gel retardation analyses, we calculate an apparent dissociation constant of 130 nM for MotA binding to the wild-type MotA box. Previous work has shown that the N-terminal region of MotA is needed for an interaction between MotA and sigma(70). We suggest that this MotA-sigma(70)interaction helps to stabilize the relatively weak interaction of MotA with the -30 region of middle promoter DNA.