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 first-shifted 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 GST-MotA 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.
Cicero
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MP
Department of Microbiology, Box 3020, Duke University Medical Center, Durham, North Carolina 27710, USA.
Alexander
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KA
Kreuzer
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KN
D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D017122:Bacteriophage T4 / Q000378:metabolism*; D001667:Binding, Competitive; D003432:Cross-Linking Reagents / Q000737:chemistry; D004274:DNA, Recombinant / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D019281:Dimerization; D005976:Glutaral / Q000737:chemistry; D011485:Protein Binding; D013050:Spectrometry, Fluorescence; D015534:Trans-Activators / Q000378:metabolism*
D001426:Bacterial Proteins; D003432:Cross-Linking Reagents; D004274:DNA, Recombinant; D004268:DNA-Binding Proteins; C063010:MotA protein, Bacteria; D015534:Trans-Activators; D005976:Glutaral