DNA Topology In TBP/DNA Complexes: Chromatin Connections

基因表达的控制对发育至关重要， 动态平衡和信号转导。在真核生物中，DNA包装成 染色质通常抑制转录。因此，转录激活 经常需要染色质重塑机械，其中两大类 组蛋白乙酰转移酶和几种类型的ATPase是破坏或 重新定位核小体。ATPase对染色质重塑的调节 对细胞周期至关重要，而组蛋白乙酰转移酶是 病毒癌蛋白。重塑的分子机制尚不清楚，但一种 共同的方面是，每个类都会从 核小体。在染色质的背景下，这预计会产生 在整个局部拓扑域中都有很大的应变。那是最近的事 研究表明，TATA盒结合蛋白(TBP)是POL II转录的中心 作为TFIID组件的一部分的启动，在 短的限制性片段的DNA环化以产生微环。 这是通过引用TBP-DNA的扁平、展开形式来解释的 很复杂。综合这些观测结果，可能存在一种间接的长程 重塑机械与TBP/TFIID：TBP IS之间的通信机制 预期与重塑的染色质结合的亲和力增加，因为 重建引起的局部拓扑展开的高自由能成本可以 在TBP-DNA复合体内通过解旋而被吸收。其效果应该是 瞬变，这是一种有选择性的生物吸引力机制 增强新修饰的染色质的转录。 目前的提案将对该模型进行批判性评估，并测试其他 前人工作的启示如下：1)DNA长度依赖于 TBP、TFIID、TBP-TFIIA等转录引起的拓扑变化 DNA环化后的因子组装将用于评估几何结构， 推定的扁平形式的灵活性和稳定性。老牌的 蒙特卡罗模拟方法将推广到这些系统。2) TBP与微环DNA结合的定量足迹实验将 用来测试预弯和预超卷曲对路径的影响 TBP与DNA结合。3)TBP突变体和TBP相关因子将用于 评估苯丙氨酸箍筋对TBP结合和拓扑的重要性， 使用DNA环化和/或微环结合试验。几个与TBP相关的 因子在转录中很重要，但它们的DNA结合特性 一直很难研究。有可能它们是有义务解开DNA的 蛋白质。4)位于含TATA DNA的5S基因上的核小体 微圆将用于直接测试重塑-绑定连接，在 体外培养。核小体和TBP之间的远程拓扑通讯是 预计会导致TBP结合的抑制。用核小体重塑 重组ISWIATPase或纯化酵母SWI/SNF可增强TBP 有约束力的。

The control of gene expression is vital to development, homeostasis, and signal transduction. In eukaryotes, DNA packaging into chromatin generally represses transcription. Thus, transcriptional activation often requires chromatin remodeling machinery, of which the two main classes are the histone acetyltransferases and several types of ATPases that disrupt or relocate nucleosomes. Chromatin remodeling by ATPases is regulated by and is essential to the cell cycle, and histone acetyltransferases are targeted by viral oncoproteins. The molecular mechanisms of remodeling are unclear, but a common aspect is that each class releases negative supercoiling from nucleosomes. In the context of chromatin, this is expected to generate substantial strain throughout the local topological domain. It was recently shown that the TATA box-binding protein (TBP), central to Pol II transcription initiation as part of the TFIID assembly, induces negative supercoiling upon DNA cyclization of short restriction fragments to give minicircies. This was explained by invoking a flattened, unwound form of the TBP-DNA complex. Combining these observations suggests a possible indirect long-range communication mechanism between the remodeling machinery and TBP/TFIID: TBP is expected to bind with increased affinity to remodeled chromatin, because the high free energy cost of local topological unwinding induced by remodeling can be absorbed by untwisting within the TBP-DNA complex. The effect should be transient, which is a biologically appealing mechanism for selectively enhancing transcription of newly-remodeled chromatin. The current proposal will critically evaluate the model and test other implications of previous work as follows: 1) The DNA length-dependence of topological changes induced by TBP, TFIID, TBP-TFIIA and other transcription factor assemblies upon DNA cyclization will be used to assess the geometry, flexibility, and stability of the putative flattened form. Well-established Monte Carlo simulation methods will be extended to these systems. 2) Quantitative footprinting experiments on TBP binding to minicircle DNA will be used to test the effect of pre-bending and pre-supercoiling on the pathway for TBP binding to DNA. 3) TBP mutants and TBP-related factors will be used to assess the importance of phenylalanine stirrups to TBP binding and topology, using DNA cyclization and/or minicircle binding assays. Several TBP-related factors are important in transcription, but their DNA binding properties have been difficult to study. It is possible that they are obligate DNA unwinding proteins. 4) A nucleosome positioned on the 5S gene in TATA-containing DNA minicircles will be used to test the remodeling-binding connection directly, in vitro. Long-range topological communication between the nucleosome and TBP is predicted to lead to repression of TBP binding. Remodeling the nucleosome with recombinant ISWI ATPase or purified yeast SWI/SNF should then potentiate TBP binding.