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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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/content/journal/micro/10.1099/00221287-147-12-3387
2001-12-01
2025-02-09
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References

  1. Aggarwal, A. K., Rodgers, D. W., Drottar, M., Ptashne, M. & Harrison, S. C. (1988). Recognition of a DNA operator by the repressor of phage 434: a view at high resolution. Science 242, 899-907.[CrossRef] [Google Scholar]
  2. Bowie, J. U. & Sauer, R. T. (1989). Identification of C-terminal extensions that protect proteins from intracellular proteolysis. J Biol Chem 264, 7596-7602. [Google Scholar]
  3. Brack, A. & Caille, A. (1978). Synthesis and beta-conformation of copolypeptides with alternating hydrophilic and hydrophobic residues. Int J Pept Protein Res 11, 128-139. [Google Scholar]
  4. Brantl, S. (1994). The copR gene product of plasmid pIP501 acts as a transcriptional repressor at the essential repR promoter. Mol Microbiol 14, 473-483.[CrossRef] [Google Scholar]
  5. Brantl, S. & Behnke, D. (1992). Copy-number control of the streptococcal plasmid pIP501 occurs at three levels. Nucleic Acids Res 20, 395-400.[CrossRef] [Google Scholar]
  6. Brantl, S. & Wagner, E. G. H. (1994). Antisense RNA-mediated transcriptional attenuation occurs faster than stable antisense/target RNA pairing: an in vitro study of plasmid pIP501. EMBO J 13, 3599-3607. [Google Scholar]
  7. Brantl, S. & Wagner, E. G. H. (1997). Dual function of the copR gene product of plasmid pIP501. J Bacteriol 179, 7016-7024. [Google Scholar]
  8. Brantl, S., Birch-Hirschfeld, E. & Behnke, D. (1993). RepR protein expression on plasmid pIP501 is controlled by an antisense RNA-mediated transcription attenuation mechanism. J Bacteriol 175, 4052-4061. [Google Scholar]
  9. Ceglowski, P. & Alonso, J. C. (1994). Gene organization of the Streptococcus pyogenes plasmid pDB101: sequence analysis of the orfη–copS region. Gene 145, 33-39.[CrossRef] [Google Scholar]
  10. Gomis-Rth, F. X., Sola, M., Acebo, P. & 7 other authors (1998). The structure of plasmid-encoded transcriptional repressor CopG unliganded and bound to its operator. EMBO J 17, 7404–7415.[CrossRef] [Google Scholar]
  11. Kuhn, K., Steinmetzer, K. & Brantl, S. (2000). Transcriptional repressor CopR: the structured acidic C terminus is important for protein stability. J Mol Biol 300, 1021-1031.[CrossRef] [Google Scholar]
  12. Milla, M. E., Brown, M. B. & Sauer, R. T. (1993). P22 Arc repressor: enhanced expression of unstable mutants by addition of polar C-terminal sequences. Protein Sci 2, 2198-2205.[CrossRef] [Google Scholar]
  13. Milla, M. E., Brown, M. B. & Sauer, R. T. (1994). Protein stability effects of a complete set of alanine substitutions in Arc repressor. Nat Struct Biol 1, 518-523.[CrossRef] [Google Scholar]
  14. Parsell, D. A. & Sauer, R. T. (1989). The structural stability of a protein is an important determinant of its proteolytic susceptibility in Escherichia coli. J Biol Chem 264, 7590-7595. [Google Scholar]
  15. Parsell, D. A., Silber, K. R. & Sauer, R. T. (1990). Carboxy-terminal determinants of intracellular protein degradation. Genes Dev 4, 277-286.[CrossRef] [Google Scholar]
  16. Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989).Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
  17. Sanger, F., Nicklen, S. & Coulson, A. R. (1977). DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74, 5463-5467.[CrossRef] [Google Scholar]
  18. Steinmetzer, K. & Brantl, S. (1997). Plasmid pIP501 encoded transcriptional repressor CopR binds asymmetrically at two consecutive major grooves of the DNA. J Mol Biol 269, 684-693.[CrossRef] [Google Scholar]
  19. Steinmetzer, K., Behlke, J. & Brantl, S. (1998). Plasmid pIP501 encoded transcriptional repressor CopR binds to its target DNA as a dimer. J Mol Biol 283, 595-603.[CrossRef] [Google Scholar]
  20. Steinmetzer, K., Hillisch, A., Behlke, J. & Brantl, S. (2000a). Transcriptional repressor CopR: structure model based localization of the DNA binding motif.Proteins 38, 393-406.[CrossRef] [Google Scholar]
  21. Steinmetzer, K., Hillisch, A., Behlke, J. & Brantl, S. (2000b). Transcriptional repressor CopR: amino acids involved in forming the dimeric interphase. Proteins 39, 408-416.[CrossRef] [Google Scholar]
  22. Swinfield, T.-J., Oultram, J. D., Thompson, E. E., Brehm, J. K. & Minton, N. P. (1990). Physical characterisation of the replication region of the Streptococcus faecalis plasmid pAMβ1. Gene 87, 79-90. [Google Scholar]
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