Graduate program in Biophysics and Structural Biology, Brandeis University, Waltham, MA, USA.
EMBO J. 2011 Aug 9;30(19):3940-6. doi: 10.1038/emboj.2011.273.
The molecular basis for regulation of lactose metabolism in Escherichia coli is well studied. Nonetheless, the physical mechanism by which the Lac repressor protein prevents transcription of the lactose promoter remains unresolved. Using multi-wavelength single-molecule fluorescence microscopy, we visualized individual complexes of fluorescently tagged RNA polymerase holoenzyme bound to promoter DNA. Quantitative analysis of the single-molecule observations, including use of a novel statistical partitioning approach, reveals highly kinetically stable binding of polymerase to two different sites on the DNA, only one of which leads to transcription. Addition of Lac repressor directly demonstrates that bound repressor prevents the formation of transcriptionally productive open promoter complexes; discrepancies in earlier studies may be attributable to transcriptionally inactive polymerase binding. The single-molecule statistical partitioning approach is broadly applicable to elucidating mechanisms of regulatory systems including those that are kinetically rather than thermodynamically controlled.
大肠杆菌中乳糖代谢调控的分子基础已得到充分研究。尽管如此，乳糖阻遏蛋白防止乳糖启动子转录的物理机制仍未解决。我们使用多波长单分子荧光显微镜，可视化了荧光标记的 RNA 聚合酶全酶与启动子 DNA 结合的单个复合物。对单分子观察的定量分析，包括使用新颖的统计分区方法，揭示了聚合酶与 DNA 上两个不同位点的高度动力学稳定结合，只有一个位点导致转录。加入 Lac 阻遏物直接证明了结合的阻遏物阻止了转录活性开放启动子复合物的形成；早期研究中的差异可能归因于转录活性聚合酶的结合。单分子统计分区方法广泛适用于阐明包括那些动力学而非热力学控制的调节系统的机制。
