Studying s 54 -dependent transcription at the single-molecule level using alternating-laser excitation (ALEX) spectroscopy
Studying s 54 -dependent transcription at the single-molecule level using alternating-laser excitation (ALEX) spectroscopy
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使用交替激光激发 (ALEX) 光谱在单分子水平研究 s 54 依赖性转录
DOI：
10.1117/12.729521
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发表时间：
2007
期刊：
影响因子：
--
通讯作者：
Heilemann M
中科院分区：
文献类型：
--
作者：
Heilemann M
我们用单分子荧光共振能量转移和交替激光激发光谱研究了σ54-dcpcndcnt基因转录复合体的单分子荧光。当时研究一个生物分子的能力使我们能够解析和分析样本的异质性，并提取关于转录的亚群和瞬时中间产物的结构信息：这些信息隐藏在批量实验中。利用位点特异性标记的σ54衍生物和SITC特异性标记的启动子片段，我们发现可以观察到单个扩散的σ54-DNA和转录起始σ“54-DNA复合体，我们可以测量这些复合体中的距离；通过凝胶迁移率改变分析证实了这些复合体的身份。我们的研究为理解σ54依赖的转录中流产起始和启动子逃逸的机制奠定了基础。
We present singlc-molcculc fluorescence studies of σ 54-dcpcndcnt gene-transcription complexes using singlemolecule fluorescence resonance energy transfer (smFRET) and alternating-laser excitation (ALEX) spectroscopy. The ability to study one biomolcculc at the time allowed us to resolve and analyze sample heterogeneities and extract structural information on subpopulations and transient intermediates of transcription: such information is hidden in bulk experiments. Using site-specifically labeled σ 54 derivatives and sitc-specificallv labeled promoter-DNA fragments, we demonstrate that we can observe single diffusing σ 54-DNA and transcription-initiation RNA polymerase-σ" 54-DNA complexes, and that we can measure distances within such complexes; the identity of the complexes has been confirmed using electrophoretic-mobility-shift assays. Our studies pave the way for understanding the mechanism of abortive initiation and promoter escape in σ 54-dependent transcription.