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首页> 外文期刊>Biochemistry >A Fluorogenic Assay To Monitor Rho-Dependent Termination of Transcription
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A Fluorogenic Assay To Monitor Rho-Dependent Termination of Transcription

机译：荧光测定以监测依赖于转录的Rho依赖性终止

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摘要

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.
机译：由环形ATP依赖性RHO电机介导的转录终止是特异性细菌的多功能调节机制，构成新抗生素的开发的有趣目标。虽然依赖性终止可以点击基因表达或有助于在给定细菌内的数百个位点处对基因组的保护，但其确切的周长和部位或物种特异性仍然存在不足。需要新的高级方法来彻底探讨RHO依赖终端的多样性以及相关机制的复杂性。电流依赖性终端的体外分析依赖于放射性标记，凝胶电泳和转录反应产物的磷磷，因此是危险的，不方便的和低通量。为了解决这些限制，我们已经使用荧光检测模态开发了第一个体外测定，以研究RHO依赖性转录终止。这种强大的实验工具在几分钟内精确地估计了终止子强度，并且针对微孔板读取器格式进行了优化，允许调用终止子序列和机制或高通量筛选靶向依赖于依赖终止的新药物的机制或高通量筛选。

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  • 来源
    《Biochemistry》 |2019年第7期|共10页
  • 作者

    Nadiras Cedric; Delaleau Mildred; Schwartz Annie; Margeat Emmanuel; Boudvillain Marc;

  • 作者单位

    CNRS UPR4301 Ctr Biophys Mol Rue Charles Sadron F-45071 Orleans 2 France;

    CNRS UPR4301 Ctr Biophys Mol Rue Charles Sadron F-45071 Orleans 2 France;

    CNRS UPR4301 Ctr Biophys Mol Rue Charles Sadron F-45071 Orleans 2 France;

    Univ Montpellier CNRS UMR 5048 INSERM U 1054 Ctr Biochim Struct 29 Rue Navacelles F-34090 Montpellier France;

    CNRS UPR4301 Ctr Biophys Mol Rue Charles Sadron F-45071 Orleans 2 France;

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  • 正文语种 eng
  • 中图分类 生物化学;
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