TraA and its N-terminal relaxase domain of the Gram-positive plasmid pIP501 show specific oriT binding and behave as dimers in solution.
TraA is the DNA relaxase encoded by the broad-host-range Grampositive 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 6xHis-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 (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 TraAN246. 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 TraAN246. 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 degrees C. CD spectra measured at 20 degrees C showed 30% a-helix and 13% b-sheet for TraA, and 27% alpha-helix and 18% beta-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.
Kopec
Jolanta
J
Fachgebiet Umweltmikrobiologie, Institut für Technischen Umweltschutz, Technische Universität Berlin, Franklinstr. 29, FR1-2, 10587 Berlin, Germany.
Bergmann
Alexander
A
Fritz
Gerhard
G
Grohmann
Elisabeth
E
Keller
Walter
W
D001483:Base Sequence; D003001:Cloning, Molecular; D004254:DNA Nucleotidyltransferases / Q000737:chemistry* / Q000378:metabolism; D004269:DNA, Bacterial / Q000737:chemistry; D019281:Dimerization; D036781:Fimbriae Proteins / Q000737:chemistry* / Q000378:metabolism; D006094:Gram-Positive Bacteria / Q000737:chemistry* / Q000235:genetics; D018933:Pili, Sex; D010957:Plasmids / Q000737:chemistry*; D011485:Protein Binding; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D014158:Transcription, Genetic
D004269:DNA, Bacterial; D036781:Fimbriae Proteins; D004254:DNA Nucleotidyltransferases; C110340:DNA relaxase