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首页> 外文期刊>Biochemistry >Sensing DNA Opening in Transcription Using Quenchable Frster Resonance Energy Transfer
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Sensing DNA Opening in Transcription Using Quenchable Frster Resonance Energy Transfer

机译：使用可淬灭的Forster共振能量转移感应转录中的DNA开放

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摘要

Many biological processes, such as gene transcription and replication, involve opening and closing ofnshort regions of double-strandedDNA (dsDNA). Few techniques, however, can study these processes in real time ornat the single-molecule level. Here, we present a F€ orster resonance energy transfer (FRET) assay that monitors thenstate ofDNA(double- vs single-stranded) at a specific region within aDNAfragment, at both the ensemble level andnthe single-molecule level. The assay utilizes two closely spaced fluorophores: a FRET donor fluorophore (Cy3B) onnthe first DNA strand and a FRET acceptor fluorophore (ATTO647N) on the complementary strand. Because ournassay is based on quenching and dequenching FRET processes, i.e., the presence or absence of contact-induced fluo-nrescence quenching, we have named it a “quenchable FRET” assay or “quFRET”. Using lac promoter DNA frag-nments, quFRET allowed us to sense transcription bubble expansion and compaction during abortive initiation bynbacterial RNA polymerase.We also used quFRET to confirm the mode of action of gp2 (a phage-encoded proteinnthat acts as a potent inhibitor ofEscherichia coli transcription) and rifampicin (an antibiotic that blocks transcriptionninitiation). Our results demonstrate that quFRET should find numerous applications in many processes involvingnDNA opening and closing, as well as in the development of new antibacterial therapies involving transcription.
机译：许多生物学过程，例如基因转录和复制，都涉及双链DNA（dsDNA）短区域的打开和关闭。但是，很少有技术可以实时研究单分子水平的这些过程。在这里，我们提出了一种F？ster共振能量转移（FRET）分析方法，该方法可以监测DNA片段内特定区域的整体DNA和单分子水平的DNA状态（双链与单链）。该测定法利用两个紧密间隔的荧光团：第一条DNA链上的FRET供体荧光团（Cy3B）和互补链上的FRET受体荧光团（ATTO647N）。因为我们的测定是基于FRET淬灭和淬灭过程，即是否存在接触诱导的荧光猝灭，所以我们将其命名为“可淬灭FRET”测定法或“ quFRET”。使用lac启动子DNA片段，quFRET使我们能够通过细菌RNA聚合酶在流产起始过程中感知转录气泡的膨胀和紧缩。转录）和利福平（一种阻止转录起始的抗生素）。我们的结果表明，quFRET应该在涉及DNA开启和关闭的许多过程以及涉及转录的新抗菌疗法的开发中找到许多应用。

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  • 来源
    《Biochemistry》 |2010年第43期|p.9171-9180|共10页
  • 作者

    Thorben Cordes Yusdi Santoso Alexandra I. Tomescu Kristofer Gryte Ling Chin Hwang Beatriz Camar Sivaramesh Wigneshweraraj and Achillefs N. Kapanidis;

  • 作者单位

    Biological Physics Research Group, Department of Physics, University of Oxford, Clarendon Laboratory, Parks Road,Oxford OX1 3PU, United Kingdom, and §Division of Microbiology, Department of Medicine, Faculty of Medicine andCentre for Molecular Microbiology and Infection, Flowers Building, Imperial College London, London SW7 2AZ,United Kingdom.) Present address: Laboratory of Molecular Biology, National Institute of Diabetes andDigestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892.;

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