Characterizing the DNA contacts and cooperative binding of F plasmid TraM to its cognate sites at oriT
Characterizing the DNA contacts and cooperative binding of F plasmid TraM to its cognate sites at oriT
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DOI：
10.1074/jbc.m111682200
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发表时间：
2002-05-10
影响因子：
4.8
通讯作者：
Frost, LS
中科院分区：
文献类型：
--
作者：
Fekete, RA;Frost, LS
TraM是一种DNA结合蛋白，用于接合转移可自我传递的IncF组质粒，包括F、R1和R100。F-TraM与F oriT中的三个位点结合。两个高亲和性结合位点sbmA和sbmB，它们是参与traM基因自动调节的几乎相同序列的直接重复;和一个低亲和性位点sbmC，一个对转移重要的反向重复，其位于最靠近转移起始的nic位点。TraM合作绑定到其结合位点在oriT; sbmA和sbmB的存在下增加了10倍的sbmC的亲和力。由TraM的oriT DNA的弯曲是最小的，这表明TraM，四聚体，能够环的DNA时，绑定到sbmA和sbmB同时。sbmA和sbmC的DNA的羟基自由基足迹显示，TraM在先前由DNase I足迹划定的区域内接触DNA。TraM保护序列CTAG内的CT碱基，其发生在sbmA的顶部和底部链上的12个碱基间隔处，与其他受保护的碱基最一致。在sbmC上的足迹显示，预测的反向重复序列被TraM保护，其模式从重复序列的中心开始，以11-12个碱基的间隔向任一链的5 '端向外辐射。在高蛋白质浓度下，这种模式延伸到DNA酶1定义的足迹之外，表明DNA包裹在蛋白质周围形成核小体样结构，这有助于准备DNA进行转移。
TraM is a DNA binding protein required for conjugative transfer of the self-transmissible IncF group of plasmids, including F, R1, and R100. F TraM binds to three sites in F oriT. two high affinity binding sites, sbmA and sbmB, which are direct repeats of nearly identical sequence involved in the autoregulation of the traM gene; and a lower affinity site, sbmC, an inverted repeat important for transfer, which is situated nearest to the nic, site where transfer originates. TraM bound cooperatively to its binding sites at oriT; the presence of sbmA and sbmB increased the affinity for sbmC 10-fold. Bending of oriT DNA by TraM was minimal, suggesting that TraM, a tetramer, was able to loop the DNA when bound to sbmA and sbmB simultaneously. Hydroxyl radical footprinting of DNA of sbmA and sbmC revealed that TraM contacted the DNA within a region previously delineated by DNase I footprinting. TraM protected the CT bases within the sequence CTAG, which occurred at 12-base intervals on the top and bottom strand of sbmA, most consistently with other protected bases. The footprint on sbmC revealed that the predicted inverted repeats were protected by TraM with a pattern that began at the center of the repeats and radiated outward at 11-12 base intervals toward the 5'-ends of either strand. At high protein concentrations, this pattern extended beyond the footprint defined by DNase 1, suggesting that the DNA was wrapped around the protein forming a nucleosome-like structure, which could aid in preparing the DNA for transfer.