Contacts between Bacillus subtilis catabolite regulatory protein CcpA and amyO target site.
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.
Kim
J H
JH
Department of Bacteriology, University of Wisconsin-Madison, E. B. Fred Hall, Madison, WI 53706, USA.
Chambliss
G H
GH
D001412:Bacillus subtilis / Q000737:chemistry* / Q000235:genetics; D001426:Bacterial Proteins; D001483:Base Sequence; D001665:Binding Sites; D019175:DNA Methylation; D004269:DNA, Bacterial / Q000737:chemistry / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D005038:Ethylnitrosourea; D005798:Genes, Bacterial; D006147:Guanine / Q000378:metabolism; D009690:Nucleic Acid Conformation; D010446:Peptide Fragments / Q000378:metabolism; D010710:Phosphates / Q000378:metabolism; D012097:Repressor Proteins / Q000378:metabolism*; D013463:Sulfuric Acid Esters; D014357:Trypsin / Q000378:metabolism; D000516:alpha-Amylases / Q000235:genetics*
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D010446:Peptide Fragments; D010710:Phosphates; D012097:Repressor Proteins; D013463:Sulfuric Acid Esters; C069286:catabolite control proteins, bacteria; D006147:Guanine; D000516:alpha-Amylases; D014357:Trypsin; C007482:dimethyl sulfate; D005038:Ethylnitrosourea