Sequence-specific interactions in the Tus-Ter complex and the effect of base pair substitutions on arrest of DNA replication in Escherichia coli
Sequence-specific interactions in the Tus-Ter complex and the effect of base pair substitutions on arrest of DNA replication in Escherichia coli
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DOI：
10.1074/jbc.272.42.26448
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发表时间：
1997-10-17
影响因子：
4.8
通讯作者：
Hill, TM
中科院分区：
文献类型：
--
作者：
CoskunAri, FF;Hill, TM
Tus蛋白和终止子（Ter）序列之间的特异性相互作用介导了大肠杆菌中DNA复制的停滞。Tus与Ter位点的结合形成了不对称的蛋白-DNA复合物，该复合物以取向依赖的方式停滞DNA复制。检测了在16个不同位置携带单碱基对取代的突变Ter位点结合纯化Tus蛋白和阻滞DNA的能力体外竞争测定表明，在位置8-19处的碱基对取代对Tus结合的自由能具有显著影响（Δ Δ G（0）为1.5至>4.0 kcal/mol）。伴随着结合亲和力的丧失，在这些位置的突变也显示出显着降低或不可检测的复制停滞活动在体内，在位置6，20，和21的取代有中度影响Tus-Ter相互作用，这表明这些碱基对有助于，但。对土族人的结合不是绝对关键的。即使对结合的影响是最小的，这些Ter突变体在阻止复制叉方面不如野生型Tus-TerB复合物有效。通过对E.大肠杆菌基因组的序列相似性的共识Ter位点序列。
Arrest of DNA replication in Escherichia coli is mediated by specific interactions between the Tus protein and terminator (Ter) sequences, Binding of Tus to a Ter site forms a asymmetric protein-DNA complex that arrests DNA replication in an orientation-dependent fashion, In this study, mutant Ter sites carrying single base pair substitutions at 16 different positions were examined for their ability to bind purified Tus protein and arrest DNA replication, In vitro competition assays demonstrated that base pair substitutions at positions 8-19 had significant effects on the free energy of Tus binding (Delta Delta G(0) Of 1.5 to >4.0 kcal/mol). Concomitant with loss of binding affinity, mutations at these positions also showed significantly lower or undetectable replication arrest activities in vivo, Substitutions at positions 6, 20, and 21 had moderate effects on Tus-Ter interactions, suggesting that these base pairs contribute to, but are. not absolutely critical for, Tus binding. Even though the effects on binding were minimal, these Ter mutants were not as efficient as wild type Tus-TerB complexes at arresting replication forks. Three new potential Ter sites, referred to as TerH, TerI, and TerJ, were identified by searching the E. coli genome for sequence similarity to a consensus Ter site sequence.