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.
Kuhn
K
K
Friedrich-Schiller-Universität Jena, Institut für Molekularbiologie, Winzerlaer Strasse 10, Jena D-07745, Germany.
Steinmetzer
K
K
Brantl
S
S
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006207:Half-Life; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics*; D017433:Protein Structure, Secondary; D012097:Repressor Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013291:Streptococcus / Q000235:genetics*; D015534:Trans-Activators / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D012097:Repressor Proteins; D015534:Trans-Activators