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首页> 外文期刊>Molecular and Cellular Biology >Spontaneous Access of Proteins to Buried Nucleosomal DNA Target Sites Occurs via a Mechanism That Is Distinct from Nucleosome Translocation
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Spontaneous Access of Proteins to Buried Nucleosomal DNA Target Sites Occurs via a Mechanism That Is Distinct from Nucleosome Translocation

机译：通过与核小体转运不同的机制，蛋白质自发地进入埋藏的核糖体DNA目标位点

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Intrinsic nucleosome dynamics termed “site exposure” provides spontaneous and cooperative access to buried regions of nucleosomal DNA in vitro. Two different mechanisms for site exposure have been proposed, one based on nucleosome translocation, the other on dynamic nucleosome conformational changes in which a stretch of the nucleosomal DNA is transiently released off the histone surface. Here we report on three experiments that distinguish between these mechanisms. One experiment investigates the effects on the accessibilities of restriction enzyme target sites inside nucleosomes when extra DNA (onto which the nucleosome may move at low energetic cost) is appended onto one end. The other two experiments test directly for nucleosome mobility under the conditions used to probe accessibility to restriction enzymes: one on a selected nonnatural nucleosome positioning sequence, the other on the well-studied 5S rRNA gene nucleosome positioning sequence. We find from all three assays that restriction enzymes gain access to sites throughout the entire length of the nucleosomal DNA without contribution from nucleosome translocation. We conclude that site exposure in nucleosomes in vitro occurs via a nucleosome conformational change that leads to transient release of a stretch of DNA from the histone surface, most likely involving progressive uncoiling from an end. Recapture at a distal site along DNA that has partially uncoiled would result in looped structures which are believed to contribute to RNA polymerase elongation and may contribute to spontaneous or ATP-driven nucleosome mobility. Transient open states may facilitate the initial entry of transcription factors and enzymes in vivo.
机译：称为“位点暴露”的内在核小体动力学提供了体外自发和协同进入核小体DNA埋藏区域的途径。已经提出了两种不同的位点暴露机制，一种基于核小体易位，另一种基于动态的核小体构象变化，其中一部分核小体DNA从组蛋白表面瞬时释放出来。在这里，我们报告了区分这些机制的三个实验。一个实验研究了将额外的DNA（核小体可以以低能量的成本移动到其上）附加到一端时，对核小体内部限制酶靶位点可及性的影响。其他两个实验在用于探测限制酶可及性的条件下直接测试了核小体的移动性：一个在选定的非天然核小体定位序列上，另一个在经过充分研究的5S rRNA基因核小体定位序列上。我们从所有三种测定中发现，限制酶可在核小体DNA的整个长度上获得对位点的访问，而不受核小体易位的影响。我们得出结论，体外核小体中的位点暴露是通过核小体构象变化发生的，该变化导致从组蛋白表面暂时释放一段DNA，最有可能涉及从末端进行的逐渐解开。沿部分解开的DNA在远端位点重新捕获将导致形成环状结构，该结构被认为有助于RNA聚合酶的延长，并可能有助于自发或ATP驱动的核小体迁移。暂态开放状态可能有助于体内转录因子和酶的初始进入。

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