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 components of plasmid pIP501. CopR binds as a dimer at two consecutive major grooves on the same face of the DNA. Previously, equilibrium dissociation constants 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 mutants were analysed with regard to activity and half-life in vivo and DNA binding, dimerization, structure and stability in vitro. The last 29 amino acid residues of CopR were not essential for DNA binding and dimerization but for protein stability. However, whereas CopDelta20 was, in spite of drastically shortened half-life, still 100 % active in vivo, CopDelta24 and CopDelta27 retained only 20 % activity. In vivo stability could be restored only partially by adding a C-terminal tail previously shown to stabilize the lambda repressor N terminus. However, substitution of seven Glu residues by Lys within the last 20 residues drastically reduced half-life. Our results clearly demonstrate that the acidic C terminus is important for the stability of CopR. Using CD-measurements we show that the C terminus of CopR is structured.
Kuhn
K
K
Friedrich-Schiller-Universität Jena, Institut für Molekularbiologie, Winzerlaer Strasse 10, Jena, D-07745, Germany.
Steinmetzer
K
K
Brantl
S
S
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D001412:Bacillus subtilis / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002942:Circular Dichroism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D019281:Dimerization; D004926:Escherichia coli; D029968:Escherichia coli Proteins; D018628:Gene Dosage; D015964:Gene Expression Regulation, Bacterial; D019791:Guanidine / Q000494:pharmacology; D006207:Half-Life; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation / Q000187:drug effects; D011489:Protein Denaturation / Q000187:drug effects; D012097:Repressor Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017384:Sequence Deletion / Q000235:genetics; D013816:Thermodynamics; D015534:Trans-Activators / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D010446:Peptide Fragments; D012097:Repressor Proteins; D015534:Trans-Activators; C489822:cueR protein, E coli; D019791:Guanidine