Integrated Graduate Program in Physical and Engineering Biology, Yale University, New Haven, CT 06520, USA; Department of Applied Physics, Yale University, New Haven, CT 06520, USA.
J Mol Biol. 2012 Nov 9;423(5):687-701. doi: 10.1016/j.jmb.2012.08.021. Epub 2012 Aug 31.
Chromatin "remodeling" is widely accepted as the mechanism that permits access to DNA by the transcription machinery. To date, however, there has been no experimental measurement of the changes in the kinetics and thermodynamics of the DNA-histone octamer association that are required to remodel chromatin so that transcription may occur. Here, we present the results of optical tweezer measurements that compare the kinetic and thermodynamic properties of nucleosomes composed of unmodified histones with those of nucleosomes that contain a mutant histone H4 (H4-R45H), which has been shown to allow SWI/SNF remodeling factor-independent transcription from the yeast HO promoter in vivo. Our measurements, carried out in a force-clamp mode, determine the force-dependent unwinding and rewinding rates of the nucleosome inner turn. At each force studied, nucleosomes containing H4-R45H unwind more rapidly and rewind more slowly than nucleosomes containing unmodified H4, indicating that the latter are the more stable. Extrapolation to forces at which the winding and unwinding rates are equal determines the absolute free energy of the nucleosome inner turn to be -32k(B)T for nucleosomes containing unmodified H4 and -27k(B)T for nucleosomes containing H4-R45H. Thus, the "loosening" or "remodeling" caused by this point mutation, which is demonstrated to be sufficient to allow transcriptional machinery access to the HO promoter (in the absence of other remodeling factors), is 5k(B)T. The correlation between the free energy of the nucleosome inner turn and the sin (SWI/SNF-independent) transcription suggests that, beyond partial unwinding, complete histone unwinding may play a role in transcriptional activation.
染色质“重塑”被广泛认为是转录机制能够接近 DNA 的机制。然而，到目前为止，还没有对重塑染色质所需的 DNA-组蛋白八聚体结合的动力学和热力学变化进行实验测量，以便转录可以发生。在这里，我们展示了光学镊子测量的结果，这些结果比较了由未修饰的组蛋白组成的核小体与含有突变组蛋白 H4（H4-R45H）的核小体的动力学和热力学性质，已证明该突变体 H4-R45H 允许 SWI/SNF 重塑因子独立地从酵母 HO 启动子进行体内转录。我们的测量是在力钳模式下进行的，确定了核小体内环的力依赖解旋和重绕速率。在研究的每个力下，含有 H4-R45H 的核小体比含有未修饰的 H4 的核小体解旋更快，重绕更慢，这表明后者更稳定。外推到缠绕和解绕速率相等的力确定了含有未修饰 H4 的核小体内环的绝对自由能为-32k(B)T，而含有 H4-R45H 的核小体的自由能为-27k(B)T。因此，这种点突变引起的“松动”或“重塑”足以允许转录机制接近 HO 启动子（在没有其他重塑因子的情况下），这是 5k(B)T。核小体内环的自由能与 sin（SWI/SNF 独立）转录之间的相关性表明，除了部分解旋之外，完整的组蛋白解旋可能在转录激活中发挥作用。
