Katrin Steinmetzer

Transcriptional repressor CopR: Structure model-based localization of the deoxyribonucleic acid binding motif

The plasmid pIP501 encoded transcriptional repressor CopR is one of the two regulators of plasmid copy number. CopR binds as a dimer to a nearly palindromic operator with the consensus sequence 5`‐CGTG. Intermediate sequence searches revealed a significant structural relationship between CopR and the bacteriophage P22 c2 and the 434 c1 repressors. In this report we describe the experimental verification of a CopR homology model, which is based on a fairly low‐sequence identity of 13.8 % to P22 c2 repressor. A model for the complex of CopR with the deoxyribonucleic acid (DNA) target was built on the basis of experimental footprinting data, the above‐mentioned CopR homology model, and the crystal structure of the 434 c1 repressor‐DNA complex. Site‐directed mutagenesis was used to test the function of amino acids involved in sequence and nonsequence‐specific DNA recognition and amino acids important for correct protein folding. CD measurements were performed to detect structural changes caused by the mutations. Exchanges of residues responsible for sequence‐specific DNA recognition reduced binding to a nonspecific level. Mutations of amino acids involved in nonspecific DNA binding lead to decreased binding affinity while maintaining selectivity. Substitution of amino acids necessary for proper folding caused dramatic structural changes. The experimental data support the model of CopR as a helix‐turn‐helix protein belonging to the λ repressor superfamily. Proteins 2000;38:393–406.

Transcriptional Repressor CopR: Amino Acids Involved in Forming the Dimeric Interface

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 KDimer revealed that the monomer of CopR is folded. Proteins 2000;39:408–416.

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

Replication of the streptococcal plasmid pIP501 is regulated by two components, CopR and the antisense RNA, RNAIII. CopR represses transcription of the essential repR mRNA about 10- to 20-fold and, additionally, prevents convergent transcription of sense and antisense RNAs. It has been demonstrated that CopR binds as a preformed dimer. DNA binding and dimerization constants were determined and amino acids were identified that are involved in DNA binding and dimerization. It was demonstrated that the C-terminal 20 aa of CopR are not involved in either activity, but play an important role for CopR stability. Furthermore, it was found that the C terminus of CopR is structured containing a beta-strand structure, most probably between the alternating hydrophilic and hydrophobic amino acids 76 and 84 (QVTLELEME). In this study stability motifs within the C terminus of CopR were dissected. Both the cognate and a heterologous (QVTVTVTVT) beta-strand structure between amino acids 76 and 84 within the C terminus stabilized CopR (CopR derivative CopVT). In contrast, substitution by a predicted alpha-helix (QVTLKLKMK) or a predicted unstructured sequence (QVTPEPEPE) caused severe and moderate destabilization, respectively. E80 seemed to be the only important C-terminal glutamic acid residue. Deletion of seven C-terminal amino acids from either wild-type CopR or CopVT reduced the half-life to approximately 50% indicating that this C-terminal sequence is a second stability motif.

Plasmid pIP501 encoded transcriptional repressor CopR: single amino acids involved in dimerization are also important for folding of the monomer.

CopR is one of the two components regulating replication of plasmid pIP501. It binds as a preformed dimer at the DNA major groove thereby repressing transcription of the essential repR-mRNA 10-20-fold. Previously, the DNA-binding motif was identified and the location of the dimeric interface was narrowed down. The C-terminal 29 residues were shown to be required exclusively for CopR stability. Here, we report the characterization of four single amino acid exchange mutants at the dimeric interface. All mutants were inactive in copy number control in vivo. Dimerization constants and DNA-binding constants were determined by analytical ultracentrifugation and EMSA, respectively. Denaturation experiments were performed to estimate the protein stability and to calculate DeltaG(0)(H(2)O). Our data indicate that the four analyzed amino acids are both involved in dimerization and proper folding of the monomer; i.e. they stabilize on the one hand the monomer and on the other hand the dimeric interface.