Boudvillain Marc, Walmacq Céline, Schwartz Annie, Jacquinot Frédérique
Centre de Biophysique Moleculaire (UPR4301), CNRS, Orleans cedex 2, Orleans, France.
Methods Mol Biol. 2010;587:137-54. doi: 10.1007/978-1-60327-355-8_10.
The transcription termination factor Rho from Escherichia coli is a ring-shaped homo-hexameric protein that preferentially interacts with naked cytosine-rich Rut (Rho utilization) regions of nascent RNA transcripts. Once bound to the RNA chain, Rho uses ATP as an energy source to produce mechanical work and disruptive forces that ultimately lead to the dissociation of the ternary transcription complex. Although transcription termination assays have been useful to study Rho activity in various experimental contexts, they do not report directly on Rho mechanisms and kinetics. Here, we describe complementary ATP-dependent RNA-DNA helicase and streptavidin displacement assays that can be used to monitor in vitro Rho's motor activity in a more direct and quantitative manner.
来自大肠杆菌的转录终止因子Rho是一种环状同型六聚体蛋白，它优先与新生RNA转录本中富含胞嘧啶的裸露Rut（Rho利用）区域相互作用。一旦与RNA链结合，Rho就利用ATP作为能量来源来产生机械功和破坏力，最终导致三元转录复合物的解离。尽管转录终止测定法在各种实验环境中对于研究Rho活性很有用，但它们并不能直接报告Rho的作用机制和动力学。在这里，我们描述了互补的ATP依赖性RNA-DNA解旋酶和链霉亲和素置换测定法，这些方法可用于以更直接和定量的方式监测体外Rho的运动活性。
