LexA-DNA bond strength by single molecule force spectroscopy
LexA-DNA bond strength by single molecule force spectroscopy
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DOI：
10.1529/biophysj.104.048868
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发表时间：
2004-10-01
影响因子：
3.4
通讯作者：
Gaub, HE
中科院分区：
文献类型：
--
作者：
Kühner, F;Costa, LT;Gaub, HE
大肠杆菌的SOS系统由两种蛋白质协调：LexA，一种由几个非连锁基因组成的阻遏蛋白，以及辅酶RecA。到目前为止，LexA控制着31个DNA结合模体略有不同的基因，允许一个基因对另一个基因进行不同程度的抑制。除了SOS系统外，LexA还在转录调控中发挥重要作用。该蛋白质通过使用特定的基序与DNA结合来调节转录，即螺旋-转弯-螺旋基序。在这里，我们使用基于AFM的单分子力谱来表征LexA蛋白与两个不同的DNA基序RecA和yebG的相互作用。我们测得recA和yebG的解离速率分别为0.045 S(-1)和0.13 S(-1)，这与LexA-recA比LexA-yebG更高的亲和力预测是一致的。结合电位宽度分别为5.4+/-1埃和4.9+/-0.5埃。这种短距离的潜力是蛋白质和DNA之间坚硬的氢键网络的特征。解绑发生在分手中，而不是渐进的滑动中。
The SOS system of Escherichia coli is coordinated by two proteins: LexA, a repressor protein of several unlinked genes, and the coprotease RecA. As known to date LexA controls 31 genes with slightly different DNA binding motifs allowing for a variable degree of repression from one gene to the other. Besides the SOS system LexA plays an important role in the regulation of transcription. The protein regulates transcription by using particular motifs to bind DNA, the helix-turn-helix motif. Here, we employed AFM-based single molecule force spectroscopy to characterize the interaction of LexA protein with two different DNA motifs: recA and yebG. We measured the dissociation rates to be 0.045 s(-1) for recA and 0.13 s(-1) for yebG, respectively, which is in accordance with the predicted higher affinity between LexA-recA compared to LexA-yebG. The widths of the binding potentials were determined to be 5.4 +/- 1 Angstrom and 4.9 +/- 0.5 Angstrom, respectively. This short-ranged potential is characteristic for a stiff hydrogen-bonding network between protein and DNA. The unbinding occurs in a breakup rather than a gradual sliding.