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. I t acts as transcriptional repressor at the essential repR promoter pII . Previously, we found that CopR contacts two consecutive major groove s (site I and site II) on the same face of the DNA. In spite of identi cal sequence motifs in these sites, neighboring bases were contacted d ifferently. 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 bind ing curve published previously is the result of two coupled equilibria : dimerization of CopR monomers and CopR dimer-DNA binding. A K-D-valu e of 1.44(+/-0.49) x 10(-6) M for CopR dimers was determined by analyt ical ultracentrifugation. Based on this value and the binding curve, t he equilibrium dissociation constant K-2 for the CopR-DNA complex was calculated to be 4(+/-1.3) x 10(-10) M. Quantitative Western blot anal ysis was used to determine the intracellular concentration of CopR in Bacillus subtilis. This value, 20 x 10(-6) to 30 x 10(-6) M, is 10 to 20-fold higher than the equilibrium constant for dimer dissociation, s uggesting that CopR binds in vivo as a preformed dimer. (C) 1998 Acade mic Press.