The MotA transcriptional activator of bacteriophage T4 binds to its specific DNA site as a monomer

During bacteriophage T4 middle mode gene expression, the MotA transcription factor binds to T4 middle promoters at a -30 mot box consensus sequence to allow activation. Previous binding studies showed that MotA forms multiple gel-shifted complexes with DNA, and structural evidence suggested that MotA dimerizes upon DNA binding. We have shown that a short (13 bp) mot box DNA substrate binds MotA protein but fails to form slower migrating complexes. Therefore, the slower migrating complexes in gel shift assays are caused by DNA-mediated binding events. Competition experiments indicate that the slower migrating complexes are formed by nonspecific binding events. While the firstshifted complex is caused by specific binding to the mot box. Saturation binding experiments revealed that the stoichiometry of MotA binding to DNA is 1:1 in the first-shifted complex, while the slower complexes apparently contain MotA multimers. Gel shift assays using mixtures of MotA and a GSTMotA fusion protein supported the conclusion that the first- shifted complex contains one protein molecule bound to DNA. Furthermore, MotA monomers were cross-linked by glutaraldehyde under conditions where slower complexes exist, but not under conditions that lead to only the first- shifted complex. We conclude that MotA binds specifically to the mot box as a monomer and that additional nonspecific binding events require flanking DNA.