Quantitative transcription factor binding kinetics at the single-molecule level.
Quantitative transcription factor binding kinetics at the single-molecule level.
复制标题
单分子水平的定量转录因子结合动力学。
DOI：
10.1016/j.bpj.2008.09.040
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发表时间：
2009
影响因子：
3.4
通讯作者：
Ong,NP
中科院分区：
文献类型：
--
作者：
Wang,Yufang;Guo,Ling;Golding,Ido;Cox,EdwardC;Ong,NP
我们用全内反射荧光显微镜研究了噬菌体λ抑制子CI与其靶DNA的结合作用。当红色荧光蛋白与单独标记的单分子DNA靶标结合和分离时，观察到与CI融合的红色荧光蛋白的强度有很大的逐步变化。用Poisson统计量描述了分子的随机缔合和解离。数以千计的个体事件被测量了暗和亮的间隔。间隔的指数分布允许直接确定缔合和解离速率常数(分别为kad和kd)。我们详细地解析了ka和kd作为三个控制参数的函数的变化：DNA长度L，CI二聚体浓度和结合亲和力。我们的结果表明，尽管与非操纵子DNA序列的相互作用是可观察到的，但CI与操纵子位点的结合并不依赖于非操纵子DNA侧翼的长度。
We investigated the binding interaction between the bacteriophageλ-repressor CI and its target DNA using total internal reflection fluorescence microscopy. Large stepwise changes in the intensity of the red fluorescent protein fused to CI were observed as it associated with and dissociated from individually labeled single-molecule DNA targets. The stochastic association and dissociation were characterized by Poisson statistics. Dark and bright intervals were measured for thousands of individual events. The exponential distribution of the intervals allowed direct determination of the association and dissociation rate constants (kaandkd, respectively). We resolved in detail howkaandkdvaried as a function of three control parameters: the DNA lengthL, the CI dimer concentration, and the binding affinity. Our results show that although interactions with nonoperator DNA sequences are observable, CI binding to the operator site is not dependent on the length of flanking nonoperator DNA.