STUDIES ON THE BINDING OF INTEGRATION HOST FACTOR (IHF) AND TRAM TO THE ORIGIN OF TRANSFER OF THE INCFV PLASMID PED208

被引:6
作者
DILAURENZIO, L
SCRABA, DG
PARANCHYCH, W
FROST, LS
机构
[1] UNIV ALBERTA,DEPT BIOL SCI,EDMONTON,AB T6G 2E9,CANADA
[2] UNIV ALBERTA,DEPT BIOCHEM,EDMONTON,AB T6G 2E9,CANADA
来源
MOLECULAR & GENERAL GENETICS | 1995年 / 247卷 / 06期
关键词
D O I
10.1007/BF00290404
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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 bp 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.
引用
收藏
页码:726 / 734
页数:9
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