Probing transcription factor dynamics at the single-molecule level in a living cell
Probing transcription factor dynamics at the single-molecule level in a living cell
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DOI：
10.1126/science.1141967
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发表时间：
2007-05-25
期刊：
影响因子：
56.9
通讯作者：
Xie, X. Sunney
中科院分区：
文献类型：
--
作者：
Elf, Johan;Li, Gene-Wei;Xie, X. Sunney
转录因子通过与DNA结合来调节基因表达。在活的大肠杆菌细胞中，我们直接观察到用荧光蛋白标记的紫胶抑制因子与染色体紫胶操作员的特异性结合。利用单分子检测技术，我们测量了抑制因子在代谢信号响应中的结合和解离动力学。此外，我们在单分子水平上表征了与DNA的非特异性结合，沿DNA片段的一维（1D）扩散以及片段之间通过细胞质的三维易位。在寻找操作符时，lac阻遏子花费大约90%的时间与DNA非特异性结合并沿DNA扩散，停留时间< 5毫秒。该方法和发现可推广到其他核酸结合蛋白。
Transcription factors regulate gene expression through their binding to DNA. In a living Escherichia coli cell, we directly observed specific binding of a lac repressor, labeled with a fluorescent protein, to a chromosomal lac operator. Using single-molecule detection techniques, we measured the kinetics of binding and dissociation of the repressor in response to metabolic signals. Furthermore, we characterized the nonspecific binding to DNA, one-dimensional (1D) diffusion along DNA segments, and 3D translocation among segments through cytoplasm at the single-molecule level. In searching for the operator, a lac repressor spends similar to 90% of time nonspecifically bound to and diffusing along DNA with a residence time of < 5 milliseconds. The methods and findings can be generalized to other nucleic acid binding proteins.