Nucleic Acids Research, Vol 25, Issue 16 3242-3247, Copyright © 1997 by Oxford University Press
C Speck, C Weigel and W Messer
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.
ARTICLES
From footprint to toeprint: a close-up of the DnaA box, the binding site for the bacterial initiator protein DnaA
Max-Planck-Institut fur Molekulare Genetik, D-14195 Berlin-Dahlem, Germany.
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