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

Friedrich-Schiller-Universität Jena, Institut für Molekularbiologie, Winzerlaer Straße 10¹, biolitec AG, Winzerlaer Straße 2a², Jena D-07745, Germany

Author for correspondence: Sabine Brantl. Tel: +49 3641 657576/78. Fax: +49 3641 657520. e-mail: Sabine.Brantl{at}rz.uni-jena.de

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 ß-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) ß-strand structure between amino acids 76 and 84 within the C terminus stabilized CopR (CopR derivative CopVT). In contrast, substitution by a predicted -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 50% indicating that this C-terminal sequence is a second stability motif.

Keywords: plasmid copy number control, circular dichroism measurements, Gram-positive bacteria, protein stability, structured C terminus

Abbreviations: CD, circular dichroism; EMSA, electrophoretic mobility-shoft assay

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				P. Freede and S. Brantl Transcriptional Repressor CopR: Use of SELEX To Study the copR Operator Indicates that Evolution Was Directed at Maximal Binding Affinity J. Bacteriol., September 15, 2004; 186(18): 6254 - 6264. [Abstract] [Full Text] [PDF]