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 de terminants within the MotA box sequence 5'TTTGCTTTA3' (positions -34 to -26 of a middle promoter) affects MotA function. Gel retardation assays with m utant MotA boxes are consistent with the idea that MotA uses minor groove c ontacts upstream and major groove contacts downstream of the center GC, and does not require any specific base feature at the CG base-pair at position -30. Ln particular, the 5-methyl residue on the thymine residue at positio n -29, a major groove contact, contributes to MotA binding, while convertin g the TA at -32 to a C . I base-pair, a change that affects the major but n or the minor groove, yields a MotA box that is similar to wild-type. Howeve r, methylation interference analyses indicate that neither the binding of M otA nor the binding of polymerase/MotA/AsiA to the middle promoter P-uvs ch i is inhibited by premethylation of guanine and adenine residues, suggestin g that binding does not require minor groove contact with any specific TA b ase-pair. Using gel retardation analyses, we calculate an apparent dissocia tion constant of 130 nM for MotA binding to the wild-type MotA box. Previou s work has shown that the N-terminal region of MotA is needed for an intera ction between MotA and sigma(70). We suggest that this MotA-sigma(70) inter action helps to stabilize the relatively weak interaction of MotA with the -30 region of middle promoter DNA. (C) 1999 Academic Press.