From footprint to toeprint: a close-up of the DnaA box, the binding site for the bacterial initiator protein DnaA.
The Escherichia coli DnaA protein binds as a monomer to the DnaA box, a 9 bp consensus sequence: 5'-TTA/TTNCACA. To assess the contribution of individual bases to protein binding we probed the DnaA-DnaA box complex with the uracil-DNA glycosylase (UDG) footprinting technique. (i) dU at the positions of T2, T4, T7' or T9' completely inhibits DnaA binding to the DnaA box. At these positions the methyl groups of the thymine residues are essential for successful DnaA binding, indicating protein contact with the major groove. Additionally they are positioned exactly on one side of the helix. (ii) dU at the position of T1 or at three T residues adjacent to the 9 bp core sequence of the DnaA box allows DnaA binding. These positions are protected from UDG digestion as revealed by the footprint assay. (iii) dU at the position of T3' on the complementary strand of teh box 5'-TTATCCACA was not protected from UDG digestion in DNA-DnaA complexes. Therefore, DnaA cannot contact the major groove at this position. In addition, a slight bend of the DnaA box towards UDG would help the enzyme to access this site.
Speck
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Max-Planck-Institut fur Molekulare Genetik, D-14195 Berlin-Dahlem, Germany.
Weigel
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Messer
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D001426:Bacterial Proteins / Q000378:metabolism*; D001665:Binding Sites; D003432:Cross-Linking Reagents; D018983:DNA Footprinting / Q000379:methods*; D045647:DNA Glycosylases; D004268:DNA-Binding Proteins / Q000378:metabolism*; D009699:N-Glycosyl Hydrolases / Q000378:metabolism*; D009690:Nucleic Acid Conformation; D013329:Structure-Activity Relationship; D051981:Uracil-DNA Glycosidase
D001426:Bacterial Proteins; D003432:Cross-Linking Reagents; D004268:DNA-Binding Proteins; C066312:DnaA protein, Bacteria; D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases; D051981:Uracil-DNA Glycosidase