Transcriptional repressor CopR: Amino acids involved in forming the dimeric interface

Plasmid pIP501 encoded transcriptional repressor CopR is one of the two reg ulators of plasmid copy number. It acts as a transcriptional repressor at t he essential repR promoter. Furthermore, CopR prevents convergent transcrip tion from the repR and the antisense promoter, thereby indirectly increasin g the amount of antisense-RNA, the second regulatory component. CopR binds as a dimer to a nearly palindromic operator with the consensus sequence 5'C GTG. Previously, a CopR structural model was built and used to identify ami no acids involved in DNA binding. These data showed that CopR is a HTH prot ein belonging to the lambda repressor superfamily and allowed the identific ation of two amino acids involved in specific DNA recognition. Here, we des cribe site-directed mutagenesis in combination with EMSA, dimerization stud ies using sedimentation equilibrium, and CD measurements to verify the mode l predictions concerning amino acids involved in dimerization. With this ap proach, the dimeric interface could be located between amino acids I44 and L62, F5 located at the N-terminus is additionally required for proper foldi ng, and could, therefore, not be unequivocally assigned to the dimeric inte rface. CD measurements at protein concentrations well below K-Dimer reveale d that the monomer of CopR is folded. (C) 2000 Wiley-Liss, Inc.