The TATA-Binding Protein Core Domain in Solution Variably Bends TATA Sequences via a Three-Step Binding Mechanism
The TATA-Binding Protein Core Domain in Solution Variably Bends TATA Sequences via a Three-Step Binding Mechanism
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DOI：
10.1021/bi8018724
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发表时间：
2009-03-03
期刊：
影响因子：
2.9
通讯作者：
Parkhurst, Lawrence J.
中科院分区：
文献类型：
--
作者：
Delgadillo, Roberto F.;Whittington, JoDell E.;Parkhurst, Lawrence J.
通过研究酵母TBP核心区与AdMLP TATA序列(TATAAAAG)的结合和弯曲，可以定量研究酵母和人TBP的N末端结构域的作用。这三种蛋白质都通过三步机制与DNA结合，没有证据表明最初结合的DNA是未弯曲的。现在，yTBP结合第一步的大活化焓和活化熵可以归因于NTD从DNA结合口袋的移动，而不是DNA弯曲的能量学。HTBP和CTBP的能量模式表明，hTBP中的158个氨基酸NTD最初并不占据DNA结合口袋。尽管hTBP和CTBP出现了相似的能量学，但在DNA结合过程中，速率常数的数量级差异导致了不同的中间体群体。我们发现，NTDS破坏了yTBP和hTBP的三种结合形式的DNA。对于这三种蛋白质，DNA弯曲角(Theta)取决于TATA序列，CTBP和hTBP的theta大于yTBP。对于这三种蛋白质，G6变体(TATAAGAG)的theta随着温度的变化而变化，并且在渗透分子存在的情况下增加类似于AdMLP。CTBP与许多变异体结合的结晶学研究表明，DNA弯曲与序列无关。这里报道的结果揭示了溶液中结合的DNA与晶体中结合的DNA在结构上的明显差异；我们将这种差异归因于晶体中渗透分子的存在。
Studies of the binding and bending of the AdMLP TATA sequence (TATAAAAG) by the core domain of yeast TBP allow quantitation of the roles of the N-terminal domains of yeast and human TBP. All three proteins bind DNA via a three-step mechanism with no evidence for an initially bound but unbent DNA. The large enthalpy and entropy of activation for the first step in yTBP binding can now be assigned to movement of the NTD from the DNA binding pocket and not to energetics of DNA bending. The energetic patterns for hTBP and cTBP suggest that the 158-amino acid NTD in hTBP does not initially occupy the DNA binding pocket. Despite the appearance of similar energetics for hTBP and cTBP, order of magnitude differences in rate constants lead to differing populations of intermediates during DNA binding. We find that the NTDs destabilize the three bound forms of DNA for both yTBP and hTBP. For all three proteins, the DNA bend angle (theta) depends on the TATA sequence, with theta for cTBP and hTBP being greater than that for yTBP. For all three proteins, theta for the G6 variant (TATAAGAG) varies with temperature and increases in the presence of osmolyte to be similar to that of AdMLP. Crystallographic studies of cTBP binding to a number of variants had shown no dependence of DNA bending on sequence. The results reported here reveal a clear structural difference for the bound DNA in solution versus the crystal; we attribute the difference to the presence of osmolytes in the crystals.