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首页> 外文期刊>Journal of Molecular Biology >An immuno-electron microscopical analysis of transcribing multinucleosomal templates: what happens to the histones?
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An immuno-electron microscopical analysis of transcribing multinucleosomal templates: what happens to the histones?

机译：转录多核小体模板的免疫电子显微镜分析：组蛋白会发生什么？

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Immuno-electron microscopy was used to visualize the structure of reconstituted chromatin after in vitro transcription by purified T7 RNA polymerase. T7 RNA polymerase disrupts the nucleosomal structure in the transcribed region. This disruption is not influenced by the template, linear or supercoiled, and the presence or absence of nucleosomal positioning sequences in the transcribed region. In this study, we used monoclonal autoantibodies reacting with the nucleosome core particles and epitopes within several regions of the four different core histones. Some of the residues recognized by the autoantibodies are accessible on the surface of the nucleosomes and some are more internal and therefore less exposed at the surface. We show that the loss of the nucleosomal configuration during transcription is due to the loss of histone/DNA binding and that at least part of the histones are transferred to the nascent RNA chains. Consequently, after in vitro transcription by T7 RNA polymerase, the nucleosomal template does not conserve its original configuration, and no interaction of antigen/antibodies is observed anymore in the region that has been transcribed. Therefore, we conclude that in our in vitro transcription assay, nucleosomes are detached from the template, and not simply unfolded with histones remaining attached to the DNA. Copyright 2000 Academic Press.
机译：免疫电子显微镜用于可视化重组T7 RNA聚合酶体外转录后的染色质结构。 T7 RNA聚合酶破坏了转录区的核小体结构。该破坏不受线性或超螺旋的模板以及转录区域中是否存在核小体定位序列的影响。在这项研究中，我们使用了单克隆自身抗体，与四个不同核心组蛋白的几个区域内的核小体核心颗粒和表位反应。自身抗体识别的某些残基可在核小体的表面上接近，而某些残基更内部，因此在表面上的暴露较少。我们表明，在转录过程中核小体构型的丧失是由于组蛋白/ DNA结合的丧失，并且至少部分组蛋白被转移到了新生的RNA链上。因此，在通过T7 RNA聚合酶进行体外转录后，核小体模板不保留其原始构型，并且在已转录的区域中不再观察到抗原/抗体的相互作用。因此，我们得出的结论是，在我们的体外转录测定中，核小体与模板分离，而不是简单地展开，而组蛋白仍与DNA相连。版权所有2000学术出版社。

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