Plasmid pIP501 encoded transcriptional repressor CopR binds to its target DNA as a dimer.
The CopR protein is one of the two regulators of pIP501 copy number. It acts as transcriptional repressor at the essential repR promoter pII. Previously, we found that CopR contacts two consecutive major grooves (site I and site II) on the same face of the DNA. In spite of identical sequence motifs in these sites, neighboring bases were contacted differently. Furthermore, we showed that CopR can dimerize in solution. We demonstrate by two independent methods that CopR binds the DNA as a dimer. We present data that suggest that the sigmoidal CopR-DNA binding curve published previously is the result of two coupled equilibria: dimerization of CopR monomers and CopR dimer-DNA binding. A KD-value of 1.44(+/-0.49)x10(-6) M for CopR dimers was determined by analytical ultracentrifugation. Based on this value and the binding curve, the equilibrium dissociation constant K2 for the CopR-DNA complex was calculated to be 4(+/-1. 3)x10(-10) M. Quantitative Western blot analysis was used to determine the intracellular concentration of CopR in Bacillus subtilis. This value, 20x10(-6) to 30x10(-6) M, is 10 to 20-fold higher than the equilibrium constant for dimer dissociation, suggesting that CopR binds in vivo as a preformed dimer.
Steinmetzer
K
K
Friedrich-Schiller-Universität Jena, Winzerlaer Str. 10, Jena, D-07745, Germany. ksteinm@imb-jena.de
Behlke
J
J
Brantl
S
S
D001412:Bacillus subtilis / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins; D001483:Base Sequence; D004269:DNA, Bacterial / Q000737:chemistry / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019281:Dimerization; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D012097:Repressor Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012689:Sequence Homology, Nucleic Acid; D015534:Trans-Activators / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014461:Ultracentrifugation
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D012097:Repressor Proteins; D015534:Trans-Activators