TraA and its N-terminal relaxase domain of the Gram-positive plasmid pIP501 show specific oriT binding and behave as dimers in solution

Jolanta Kopec, Alexander Bergmann, Gerhard Fritz, Elisabeth Grohmann, Walter Keller*

*Corresponding author for this work

Research output: Contribution to journalArticle

Abstract

TraA is the DNA relaxase encoded by the broad-host-range Gram-positive plasmid pIP501. It is the second relaxase to be characterized from plasmids originating from Gram-positive organisms. Full-length TraA (654 amino acids) and the N-terminal domain (246 amino acids), termed TraAN246, were expressed as 6 x His-tagged fusions and purified. Small-angle X-ray scattering and chemical cross-linking proved that TYaAN246 and TraA form dimers in solution. Both proteins revealed oriTpIP501 (origin of transfer of pIP501) cleavage activity on supercoiled plasmid DNA in vitro. oriT binding was demonstrated by electrophoretic mobility shift assays. Radiolabelled oligonucleotides covering different parts of OriTpIP501 were subjected to binding with TraA and TmAN246. The KD of the protein-DNA complex encompassing the inverted repeat, the nick site and an additional 7 bases was found to be 55 nM for TraA and 26 nM for TraAN 246. The unfolding of both protein constructs was monitored by measuring the change in the CD signal at 220 nm upon temperature change. The unfolding transition of both proteins occurred at approx. 42°C. CD spectra measured at 20°C showed 30% α-helix and 13% β-sheet for TraA, and 27% α-helix and 18% β-sheet content for the truncated protein. Upon DNA binding, an enhanced secondary structure content and increased thermal stability were observed for the TraAN246 protein, suggesting an induced-fit mechanism for the formation of the specific relaxase-oriT complex.

Original languageEnglish
Pages (from-to)401-409
Number of pages9
JournalBiochemical Journal
Volume387
Issue number2
DOIs
Publication statusPublished - 15 Apr 2005

Keywords

  • Aggregation state
  • Circular dichroism
  • DNA-protein interaction
  • Plasmid pIP501
  • Relaxase
  • Small-angle X-ray scattering

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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