Zhang Zhengjian, Revyakin Andrey, Grimm Jonathan B, Lavis Luke D, Tjian Robert
Janelia Farm Research Campus, Howard Hughes Medical Institute, Ashburn, United States.
Elife. 2014;3:e01775. doi: 10.7554/eLife.01775. Epub 2014 Jan 28.
Transcription is an inherently stochastic, noisy, and multi-step process, in which fluctuations at every step can cause variations in RNA synthesis, and affect physiology and differentiation decisions in otherwise identical cells. However, it has been an experimental challenge to directly link the stochastic events at the promoter to transcript production. Here we established a fast fluorescence in situ hybridization (fastFISH) method that takes advantage of intrinsically unstructured nucleic acid sequences to achieve exceptionally fast rates of specific hybridization (∼10e7 M(-1)s(-1)), and allows deterministic detection of single nascent transcripts. Using a prototypical RNA polymerase, we demonstrated the use of fastFISH to measure the kinetic rates of promoter escape, elongation, and termination in one assay at the single-molecule level, at sub-second temporal resolution. The principles of fastFISH design can be used to study stochasticity in gene regulation, to select targets for gene silencing, and to design nucleic acid nanostructures. DOI: http://dx.doi.org/10.7554/eLife.01775.001.
转录是一个本质上随机、有噪声且多步骤的过程，其中每一步的波动都会导致RNA合成的变化，并影响原本相同细胞中的生理和分化决定。然而，将启动子处的随机事件与转录本产生直接联系起来一直是一项实验挑战。在这里，我们建立了一种快速荧光原位杂交（fastFISH）方法，该方法利用内在无序的核酸序列实现了异常快速的特异性杂交速率（约10e7 M(-1)s(-1)），并允许对单个新生转录本进行确定性检测。使用一种典型的RNA聚合酶，我们展示了在单分子水平上利用fastFISH在一次测定中以亚秒级时间分辨率测量启动子逃逸、延伸和终止的动力学速率。fastFISH设计的原理可用于研究基因调控中的随机性、选择基因沉默的靶点以及设计核酸纳米结构。DOI: http://dx.doi.org/10.7554/eLife.01775.001
