Transcriptional repressor CopR: the structured acidic C terminus is important for protein stability

The transcriptional repressor CopR is one of the two copy-number control co mponents of plasmid pIP501. CopR binds as a dimer at two consecutive major grooves on the same face of the DNA. Previously, equilibrium dissociation c onstants of CopR dimers and the CopR-DNA complex and the intracellular CopR concentration were calculated. Amino acid residues involved in DNA binding and dimerization were determined. Here, we provide a detailed analysis of the acidic C terminus of CopR. A series of C-terminally truncated CopR muta nts were analysed with regard to activity and half-life in vivo and DNA bin ding, dimerization, structure and stability in vitro. The last 29 amino aci d residues of CopR were not essential for DNA binding and dimerization but for protein stability. However, whereas Cop Delta 20 was, in spite of drast ically shortened half-life, still 100% active in vivo, Cop Delta 24 and Cop Delta 27 retained only 20% activity. In vivo stability could be restored o nly partially by adding a C-terminal tail previously shown to stabilize the lambda repressor N terminus. However, substitution of seven Glu residues b y Lys within the last 20 residues drastically reduced half-life. Our result s clearly demonstrate that the acidic C terminus is important for the stabi lity of CopR. Using CD-measurements we show that the C terminus of CopR is structured. (C) 2000 Academic Press.