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首页> 外文期刊>Journal of Molecular Biology >TraY DNA Recognition of Its Two F Factor Binding Sites.
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TraY DNA Recognition of Its Two F Factor Binding Sites.

机译：TraY DNA识别其两个F因子结合位点。

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摘要

F factor TraY, a ribbon-helix-helix DNA-binding protein, performs two roles in bacterial conjugation. TraY binds the F origin of transfer (oriT) to promote nicking of plasmid DNA prior to conjugative transfer. TraY also binds the P(Y) promoter to up-regulate tra gene expression. The two plasmid regions bound by TraY share limited sequence identity, yet TraY binds them with similar affinities. TraY recognition of the two sites was first probed using in vitro footprinting methods. Hydroxyl radical footprinting at both oriT and P(Y) sites indicated that bound TraY protected the DNA backbone bordering three adjacent DNA subsites. Analytical ultracentrifugation results for TraY:oligonucleotide complexes were consistent with two of these subsites being bound cooperatively, and the third being occupied at higher TraY concentrations. Methylation protection and interference footprinting identified several guanine bases contacted by or proximal to bound TraY, most located within these subsites. TraY affinity for variant oriT sequences with base substitutions at or near these guanine bases suggested that two of the three subsites correspond to high-affinity, cooperatively bound imperfect inverted GA(G/T)A repeats. Altering the spacing or orientation of these sites reduced binding. TraY mutant R73A failed to protect two symmetry-related oriT guanine bases in these repeats from methylation, identifying possible direct TraY-DNA contacts. The third subsite appears to be oriented as an imperfect direct repeat with its adjacent subsite, although base substitutions at this subsite did not reduce binding. Although unusual for ribbon-helix-helix proteins, this binding site arrangement occurs at both F TraY sites, consistent with it being functionally relevant.
机译：F因子TraY是一种带状-螺旋-螺旋DNA结合蛋白，在细菌结合中起两个作用。 TraY结合F转移起点（oriT），以促进共轭转移之前质粒DNA的切口。 TraY还与P（Y）启动子结合，从而上调tra基因的表达。 TraY结合的两个质粒区域共享有限的序列同一性，但TraY以相似的亲和力结合它们。首先使用体外足迹法探测两个站点的TraY识别。在oriT和P（Y）位点的羟基自由基足迹表明，结合的TraY保护与三个相邻DNA亚位点接壤的DNA主链。 TraY：寡核苷酸复合物的超离心分析结果与其中两个亚位点协同结合一致，而第三个位点在较高的TraY浓度下一致。甲基化保护和干扰足迹识别了与结合的TraY接触或邻近的鸟嘌呤碱基，大多数位于这些亚位点。对在这些鸟嘌呤碱基处或附近具有碱基取代的oriT变异序列的TraY亲和力表明，这三个子位点中的两个对应于高亲和力，合作结合的不完全反向GA（G / T）A重复序列。改变这些位点的间隔或方向减少了结合。 TraY突变体R73A无法保护这些重复序列中的两个对称相关oriT鸟嘌呤碱基免受甲基化，从而确定了可能的TraY-DNA直接接触。第三个亚位点似乎是与其相邻亚位点的不完全直接重复，尽管在该亚位点的碱基取代并没有减少结合。尽管对于带状-螺旋-螺旋蛋白不常见，但是这种结合位点排列发生在两个F TraY位点上，这与它在功能上是相关的一致。

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