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Characterization of the oriT region of the IncFV plasmid pED208 (1991)

Laurenzio, L. ; Frost, L. S. ; Finlay, B. B. ; [et al.]

Oxford, UK : Blackwell Publishing Ltd

Molecular microbiology 5 (1991), S. 0

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ISSN: 1365-2958

Source: Blackwell Publishing Journal Backfiles 1879-2005

Topics: Biology , Medicine

Notes: DNA sequence analysis of a 2.2kb EcoRI-Hin dIII fragment from pED208, the derepressed form of the IncFV plasmid Folac, revealed sequences highly homologous to the oriT region, traM, and traJ genes of other IncF plasm ids. The TraM protein was purified and immunoblots of fractionated cells containing pED208 or Folac showed that TraM was predominantly in the cytoplasm. Using DNA retardation assays and the DNase I footprinting technique, the TraM protein was found to bind to three large motifs in the oriT region: (I) an inverted repeat, (II) two direct repeats, and (III) the traM promoter region. These three footprint regions contained a HinfI-like sequence (GANTC) that appeared 16 times, spaced 11–12bp (or multiples thereof) apart, suggesting that TraM protein binds in a complex manner over this entire region.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1111/j.1365-2958.1991.tb01927.x

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Studies on the binding of integration host factor (IHF) and TraM to the origin of transfer of the IncFV plasmid pED208 (1995)

Laurenzio, L. ; Scraba, D. G. ; Paranchych, W. ; [et al.]

Springer

Molecular genetics and genomics 247 (1995), S. 726-734

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ISSN: 1617-4623

Source: Springer Online Journal Archives 1860-2000

Topics: Biology

Notes: Abstract The origin of transfer (oriT) of the IncFV plasmid pED208 contains a region with three binding sites for both the plasmid-encoded TraM protein and the integration host factor (IHF) of Escherichia coli, a sequence-specific DNA-binding protein. One region, containing overlapping TraM and IHF binding sites, could be interpreted as containing two binding sites for each protein. Using gel retardation assays, an affinity constant for IHF binding to the three main sites was estimated in the presence and absence of 0.1 M potassium glutamate, which increased the avidity of IHF binding to the weaker sites by two orders of magnitude. DNase I protection analyses and electron microscopy were used to determine the affinity of IHF for oriT-containing DNA in the presence and absence of TraM. The binding of IHF and TraM was found to be non-cooperative by the two techniques employed. Electron microscopy also demonstrated that IHF bent the oriT region in a manner consistent with its previously determined mode of action, while TraM had no discernible effect on the appearance of the DNA. This suggested that IHF and TraM interact with a 295 by sequence in the oriT region and organize it into a higher order structure that may have a role in the initiation of DNA transfer and control of traM expression.