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

Abstract

Plasmid pIP501 encoded transcriptional repressor CopR is one of the two regulators of plasmid copy number. It acts as a transcriptional repressor at the essential repR promoter. Furthermore, CopR prevents convergent transcription from the repR and the antisense promoter, thereby indirectly increasing the amount of antisense-RNA, the second regulatory component. CopR binds as a dimer to a nearly palindromic operator with the consensus sequence 5'CGTG. Previously, a CopR structural model was built and used to identify amino acids involved in DNA binding. These data showed that CopR is a HTH protein belonging to the lambda repressor superfamily and allowed the identification of two amino acids involved in specific DNA recognition. Here, we describe site-directed mutagenesis in combination with EMSA, dimerization studies using sedimentation equilibrium, and CD measurements to verify the model predictions concerning amino acids involved in dimerization. With this approach, the dimeric interface could be located between amino acids I44 and L62. F5 located at the N-terminus is additionally required for proper folding, and could, therefore, not be unequivocally assigned to the dimeric interface. CD measurements at protein concentrations well below K(Dimer) revealed that the monomer of CopR is folded.

Equilibrium denaturation studies of the Escherichia coli factor for inversion stimulation: Implications for in vivo function

“…The ability to measure dimer and monomer populations using serially diluted FIS samples should allow for the calculation of the dimer dissociation constant ( K d ). Although this is ideally accomplished using a method that directly monitors quaternary structure, CD can be used to measure a protein's K d if the protein unfolds via a two‐state mechanism (Azuma and Hamaguchi 1976; Franchini and Reid 1999; Steinmetzer et al 2000). In this case, the disappearance of the CD signal should occur in parallel with subunit dissociation.…”

Transcriptional repressor CopR: dissection of stabilizing motifs within the C terminus

“…The resulting 500 bp PCR fragment was digested with BamHI and inserted into the single BamHI site of plasmid pPR7 (Brantl & Behnke, 1992). For the construction of plasmid pCOPEVT, the 500 bp BamHI fragment encoding CopVT was isolated from plasmid pCOPVT and inserted into the single BamHI site of plasmid pPR1-E (Steinmetzer et al, 2000a).…”

“…A single-step PCR with primer B618-30 (Steinmetzer & Brantl, 1997) and (for pQVT) primer 951-31 (Steinmetzer et al, 2000a) or (for pQPE) primer 8 (Table 1) was used to amplify the mutated copR genes from the E. coli\B. subtilis shuttle vectors pCOPVT and pCOPPE used as templates.…”

Transcriptional repressor CopR acts by inhibiting RNA polymerase binding

“…Furthermore, we demonstrated that CopR binds exclusively as a dimer and calculated the equilibrium dissociation constants for the CopR dimers and the CopR-DNA complex to be 0.4 nM and 1.4 mM, respectively (Steinmetzer et al, 1998). A 3D model of the N-terminal 63 amino acids of CopR was built, allowing the identification of amino acids involved in DNA binding and dimerization (Steinmetzer et al, 2000a(Steinmetzer et al, , b, 2002a. Furthermore, it was established that the structured acidic C terminus of CopR that forms a b-strand is necessary for stabilization of the protein (Kuhn et al, 2000(Kuhn et al, , 2001.…”