Protacio R U, Polach K J, Widom J
Department of Biochemistry Molecular Biology, and Cell Biology, Northwestern University, Evanston, IL 60208-3500, USA.
J Mol Biol. 1997 Dec 19;274(5):708-21. doi: 10.1006/jmbi.1997.1440.
The packaging of DNA in nucleosomes presents obstacles to the action of gene regulatory proteins and polymerases on their natural chromatin substrates. We recently reported that nucleosomes exist in a conformational equilibrium, transiently exposing stretches of their DNA off the histone surface. Such "site exposure" processes potentially provide the needed access of proteins to DNA in chromatin. However, the experiments that reveal site exposure are carried out on timescales of tens of minutes to hours. The actual rates of site exposure are not known. Here we use T7 RNA polymerase and exonuclease III as probes to obtain a more relevant lower bound on the rate of nucleosomal site exposure. We find that the organization of DNA into nucleosomes detectably slows the elongation rate of the polymerase, but that full-length elongation, which requires access to all of the DNA, occurs on the seconds timescale. Independent experiments with exonuclease III, which probes the outermost DNA segments only, similarly show that site exposure in these regions occurs on a timescale of seconds or faster. We conclude that site exposure is sufficiently rapid that it may play a role in the initial binding of regulatory proteins to nucleosomal target sites. These rapid rates argue against a nucleosome sliding model for the mechanism of site exposure. Surprisingly, the measured rates may be too slow to account for the known rates of polymerase elongation in vivo. Mechanisms by which polymerase progression through nucleosomes might be catalyzed are discussed.
DNA在核小体中的包装对基因调控蛋白和聚合酶作用于其天然染色质底物构成了障碍。我们最近报道，核小体存在构象平衡，会短暂地将其DNA片段暴露于组蛋白表面之外。这种“位点暴露”过程可能为蛋白质提供了在染色质中接触DNA所需的途径。然而，揭示位点暴露的实验是在几十分钟到几小时的时间尺度上进行的。实际的位点暴露速率尚不清楚。在这里，我们使用T7 RNA聚合酶和核酸外切酶III作为探针，以获得关于核小体位点暴露速率更相关的下限。我们发现，DNA组装成核小体会显著减慢聚合酶的延伸速率，但需要接触所有DNA的全长延伸在数秒的时间尺度上发生。仅探测最外层DNA片段的核酸外切酶III的独立实验同样表明，这些区域的位点暴露在数秒或更快的时间尺度上发生。我们得出结论，位点暴露足够迅速，可能在调控蛋白与核小体靶位点的初始结合中发挥作用。这些快速速率与位点暴露机制的核小体滑动模型相悖。令人惊讶的是，测得速率可能太慢，无法解释体内已知的聚合酶延伸速率。文中讨论了聚合酶通过核小体进行延伸可能被催化的机制。
