Centre de Biophysique Moléculaire , CNRS UPR4301 , rue Charles Sadron , 45071 Orléans cedex 2, France.
ED 549, Sciences Biologiques & Chimie du Vivant , Université d'Orléans , 45100 Orléans , France.
Biochemistry. 2019 Feb 19;58(7):865-874. doi: 10.1021/acs.biochem.8b01216. Epub 2019 Jan 24.
Transcription termination mediated by the ring-shaped, ATP-dependent Rho motor is a multipurpose regulatory mechanism specific to bacteria and constitutes an interesting target for the development of new antibiotics. Although Rho-dependent termination can punctuate gene expression or contribute to the protection of the genome at hundreds of sites within a given bacterium, its exact perimeter and site- or species-specific features remain insufficiently characterized. New advanced approaches are required to explore thoroughly the diversity of Rho-dependent terminators and the complexity of associated mechanisms. Current in vitro analyses of Rho-dependent termination rely on radiolabeling, gel electrophoresis, and phosphorimaging of transcription reaction products and are thus hazardous, inconvenient, and low-throughput. To address these limitations, we have developed the first in vitro assay using a fluorescence detection modality to study Rho-dependent transcription termination. This powerful experimental tool accurately estimates terminator strengths in a matter of minutes and is optimized for a microplate reader format allowing multiplexed characterization of putative terminator sequences and mechanisms or high-throughput screening of new drugs targeting Rho-dependent termination.
由环形、ATP 依赖性 Rho 分子马达介导的转录终止是一种细菌特有的多用途调控机制，是开发新型抗生素的一个有趣靶点。虽然 Rho 依赖性终止可以在给定细菌内的数百个位点上打断基因表达或有助于保护基因组，但它的确切边界和位点或物种特异性特征仍未得到充分描述。需要新的先进方法来彻底探索 Rho 依赖性终止子的多样性和相关机制的复杂性。目前，体外分析 Rho 依赖性终止依赖于放射性标记、凝胶电泳和转录反应产物的磷成像，因此具有危险性、不方便和低通量的特点。为了解决这些限制，我们开发了第一个使用荧光检测模式的体外测定法来研究 Rho 依赖性转录终止。这种强大的实验工具可以在几分钟内准确估计终止子的强度，并针对微孔板读取器格式进行了优化，允许对潜在终止子序列和机制进行多路复用表征，或对靶向 Rho 依赖性终止的新型药物进行高通量筛选。
