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Volume 147, Issue 12

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

Abstract

Replication of the streptococcal plasmid pIP501 is regulated by two components, CopR and the antisense RNA, RNAIII. CopR represses transcription of the essential 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 β-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) β-strand structure between amino acids 76 and 84 within the C terminus stabilized CopR (CopR derivative CopVT). In contrast, substitution by a predicted α-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.

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  • Published Online:
Keyword(s): CD, circular dichroism , circular dichroism measurements , EMSA, electrophoretic mobility-shoft assay , Gram-positive bacteria , plasmid copy number control , protein stability and structured C terminus
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2001-12-01
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Transcriptional repressor CopR: dissection of stabilizing motifs within the C terminus
Microbiology 147, 3387 (2001); https://doi.org/10.1099/00221287-147-12-3387
/content/journal/micro/10.1099/00221287-147-12-3387
/content/journal/micro/10.1099/00221287-147-12-3387
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