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 K(Dimer) revealed that the monomer of CopR is folded.
Steinmetzer
K
K
Institut für Molekularbiologie, Friedrich-Schiller-Universität Jena, Jena, Germany.
Hillisch
A
A
Behlke
J
J
Brantl
S
S
D000595:Amino Acid Sequence; D000596:Amino Acids; D001412:Bacillus subtilis; D001426:Bacterial Proteins; D002942:Circular Dichroism; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019281:Dimerization; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary; D012097:Repressor Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015534:Trans-Activators / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic
D000596:Amino Acids; D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D012097:Repressor Proteins; D015534:Trans-Activators; D004247:DNA