DNA Topology In TBP/DNA Complexes: Chromatin Connections

基因表达的控制对发育至关重要， 稳态和信号转导。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 通常需要染色质重塑机制，其中两个主要类别 是组蛋白乙酰转移酶和几种类型的ATP酶， 重新定位核小体。ATP酶对染色质的重塑受 细胞周期所必需的，组蛋白乙酰转移酶是靶向的， 病毒癌蛋白重塑的分子机制尚不清楚，但 一个共同的方面是，每一类都从 核小体在染色质的背景下，这有望产生 在整个局部拓扑结构域中的大量应变。最近有 显示TATA盒结合蛋白（TBP），Pol II转录的中心， 作为TFIID组装的一部分，启动诱导负超螺旋， 短限制性片段的DNA环化以产生微环。 这是通过调用TBP-DNA的扁平解旋形式来解释的 复杂.结合这些观察结果表明， 重塑机制和TBP/TFIID之间的通信机制：TBP是 预期与重塑染色质的亲和力增加，因为 由重构引起局部拓扑解旋的高自由能代价可 通过在TBP-DNA复合物内解旋而被吸收。效果应该 短暂的，这是一种生物学上有吸引力的机制， 增强新重塑的染色质的转录。 目前的建议将严格评估模型和测试其他 研究结果表明：（1）DNA的长度依赖性 TBP、TFIID、TBP-TFIIA和其他转录诱导的拓扑变化 DNA环化后的因子组装将用于评估几何形状， 灵活性和稳定性的假定扁平形式。完善的 蒙特卡罗模拟方法将扩展到这些系统。（二） TBP与微环DNA结合的定量足迹实验将在 用于测试预弯曲和预超盘绕对以下路径的影响： TBP与DNA结合。3)TBP突变体和TBP相关因子将用于 评估苯丙氨酸对TBP结合和拓扑结构的重要性， 使用DNA环化和/或微环结合测定。几个TBP相关 因子在转录中很重要，但它们的DNA结合特性 很难研究。它们可能是专性DNA解旋 proteins. 4)含TATA的DNA中位于5S基因上的核小体 微环将用于直接测试重塑绑定连接， 体外核小体和TBP之间的远程拓扑通讯是 预测导致TBP结合的抑制。核小体的重塑 重组ISWI ATP酶或纯化的酵母SWI/SNF则应增强TBP 约束力

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.