Nucleic Acids Research, Vol 25, Issue 17 3490-3496, Copyright © 1997 by Oxford University Press
JH Kim and GH Chambliss
Catabolite control protein A (CcpA) is a global regulatory protein involved
in catabolite repression and glucose activation in Gram- positive bacteria.
cis -Acting DNA sequences, catabolite response elements ( cre s), involved
in this regulatory system contain a 14 base pair (bp) region of dyad
symmetry. CcpA, a repressor of the Lac I family, has been shown to bind
specifically to cre s. To better understand cre recognition by CcpA, we
have focused on the interaction between CcpA and the amyE cre , called
amyO, which is located at the transcription start site of the alpha-amylase
gene. DNA-protein complexes were probed with dimethylsulfate (DMS) and N -
ethylnitrosourea (EtNU) to identify guanines and phosphates that
participate in complex formation. Interaction between amyO and CcpA
visualized through methylation protection and interference showed that CcpA
contacts guanine residues at the outer bounds of amyO with higher affinity
than near the dyad axis. From ethylation interference studies, it was found
that CcpA contacts three phosphate groups at each end of amyO, and one or
two phosphate groups near the dyad axis. Exonuclease III protection
revealed that CcpA protects a 26 bp region centered around the dyad axis of
amyO. The isolated N-terminal fragment still specifically bound to the
sequence resembling the half sites of the amyO sequence. Considering these
findings and the helical structure of B-DNA, our results suggest that each
of the two monomers of the CcpA molecule contact the major groove in each
half of the region of dyad symmetry and that the contacts are on the same
face of the DNA helix, which is typical of bacterial repressor-operator
interactions. However, the absence of strong contacts near the dyad axis by
CcpA is in contrast to the situation with the gal repressor, another member
of the Lac I family of repressors.
ARTICLES
Contacts between Bacillus subtilis catabolite regulatory protein CcpA and amyO target site
Department of Bacteriology, University of Wisconsin-Madison, E. B. Fred Hall, Madison, WI 53706, USA.
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