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Structure analysis of DNA relaxases, the key enzymes of bacterial conjugation: TraA and its N-terminal relaxase domain of the Gram-positive plasmid pIP501 show specific oriT binding and behave as dimers in solution
Zitatschlüssel Kopec2006
Autor Kopec, Jolanta
Jahr 2006
Schule Technische Universität Berlin, FG Umweltmikrobiologie
Zusammenfassung TraA is the DNA relaxase encoded by the broad-host-range Gram-positive plasmid pIP501. It is the second relaxase characterized from plasmids originating from Gram-positive organisms. TraA (654 amino acids) and the N-terminal domain (246 amino acids), termed TraAN246, were expressed as 6×His-tagged fusions and purified. Small-angle X-ray scattering and chemical cross-linking proved that TraAN246 and TraA form dimers in solution. Both proteins revealed oriTpIP501 cleavage activity on supercoiled plasmid DNA in vitro. oriT binding was demonstrated by electrophoretic mobility shift assays. Radiolabeled oligonucleotides covering different parts of oriTpIP501 were subjected to binding with TraA and TraAN246. The KD of the protein-DNA complex encompassing the inverted repeat (IR), the nick site and additional 7 bases, was found to be 55 nM for TraA, and 26 nM for TraAN246. The unfolding of both protein constructs was monitored by measuring the change in the circular dichroism (CD) signal at 220 nm upon temperature change. The unfolding transition of both proteins occurred at around 42°C. CD spectra measured at 20°C showed 30 % ?-helix and 13 % ?-sheet for TraA and 27 % ?-helix and 18 % ?-sheet content for the truncated protein. Upon DNA binding an enhanced secondary structure content and increased thermal stability was observed for the TraAN246 protein suggesting an induced-fit mechanism for the formation of the specific relaxase-oriT complex. Both proteins bind to promoter region of tra operon and therefore regulate its transcription. Expression of soluble Orf7 protein was achieved, the protein turned out to possess lytic transglycosylase (LT) activity. 3D structure models were calculated for Orf10 and LT domain of Orf7. These models can be used as search models in crystal structure solution by molecular replacement method.
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