Transcriptional repressor CcpN from Bacillus subtilis compensates asymmetric contact distribution by cooperative binding.
Carbon catabolite repression in Bacillus subtilis is carried out mainly by the major regulator CcpA. In contrast, sugar-dependent repression of three genes, sr1 encoding a small untranslated RNA, and two genes, gapB and pckA, coding for gluconeogenic enzymes is mediated by the recently identified transcriptional repressor CcpN. Since previous DNase I footprinting yielded only basic information on the operator sequences of CcpN, chemical interference footprinting studies were performed for a precise contact mapping. Methylation interference, potassium permanganate and hydroxylamine footprinting were used to identify all contacted residues in both strands in the three operator sequences. Furthermore, ethylation interference experiments were performed to identify phosphate residues essential for CcpN binding. Here, we show that each operator has two binding sites for CcpN, one of which was always contacted more strongly than the other. The three sites that exhibited close contacts were very similar in sequence, with only a few slight variations, whereas the other three corresponding sites showed several deviations. Gel retardation assays with purified CcpN demonstrated that the differences in contact number and strength correlated well with significantly different K(D) values for the corresponding single binding sites. However, quantitative DNase I footprinting of whole operator sequences revealed cooperative binding of CcpN that, apparently, compensated the asymmetric contact distribution. Based on these data, possible consequences for the repression mechanism of CcpN are discussed.
Licht
Andreas
A
AG Bakteriengenetik, Friedrich-Schiller-Universität Jena, Philosophenweg 12, D-07743 Jena, Germany. andreas.licht@uni-jena.de
Brantl
Sabine
S
D001426:Bacterial Proteins / Q000737:chemistry*; D001483:Base Sequence; D001665:Binding Sites; D018983:DNA Footprinting; D004268:DNA-Binding Proteins / Q000737:chemistry*; D003850:Deoxyribonuclease I / Q000737:chemistry; D019811:Hydroxylamine / Q000737:chemistry; D008745:Methylation; D008969:Molecular Sequence Data; D009875:Operator Regions, Genetic; D011196:Potassium Permanganate / Q000737:chemistry; D012097:Repressor Proteins / Q000737:chemistry*
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D012097:Repressor Proteins; C069286:catabolite control proteins, bacteria; D011196:Potassium Permanganate; D019811:Hydroxylamine; D003850:Deoxyribonuclease I