Backtracking determines the force sensitivity of RNAP II in a factor-dependent manner
Backtracking determines the force sensitivity of RNAP II in a factor-dependent manner
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DOI：
10.1038/nature05701
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发表时间：
2007-04-12
期刊：
影响因子：
64.8
通讯作者：
Bustamante, Carlos
中科院分区：
文献类型：
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作者：
Galburt, Eric A.;Grill, Stephan W.;Bustamante, Carlos
RNA聚合酶II（RNAP II）负责在从酵母到人类1的高度调节的过程中转录真核细胞中的所有信使RNA，并且作为细胞功能的中心控制点。在这里，我们研究了来自酿酒酵母的单个RNAP II分子在存在和不存在TFIIS的情况下的转录动力学，TFIIS是一种已知通过核小体屏障增加转录的转录延伸因子（2\）。使用单分子双阱光镊分析结合一种新的方法来富集活性复合物，我们发现RNAP II对阻碍力的响应完全由酶回溯决定（3-6）。令人惊讶的是，RNAP II分子在7.5 +/- 2 pN的力下停止转录，并且无法从回溯中恢复，该力仅为大肠杆菌RNAP测定的力的三分之一（7，8）。我们发现，回溯暂停持续时间遵循t（-3/2）的幂律，这意味着在回溯RNAP II扩散离散的碱基对步骤，并表明回溯可能占大多数RNAP II暂停。值得注意的是，TFIIS的加入挽救了回溯酶，并允许转录进行到16.9 +/- 3.4 pN的力。总之，这些结果描述了真核生物中转录延伸的调节机制，通过该机制，转录因子改变RNAP II的机械性能，使其能够对抗更高的负载。
RNA polymerase II ( RNAP II) is responsible for transcribing all messenger RNAs in eukaryotic cells during a highly regulated process that is conserved from yeast to human 1, and that serves as a central control point for cellular function. Here we investigate the transcription dynamics of single RNAP II molecules from Saccharomyces cerevisiae against force and in the presence and absence of TFIIS, a transcription elongation factor known to increase transcription through nucleosomal barriers(2\). Using a single-molecule dual-trap optical-tweezers assay combined with a novel method to enrich for active complexes, we found that the response of RNAP II to a hindering force is entirely determined by enzyme backtracking(3-6). Surprisingly, RNAP II molecules ceased to transcribe and were unable to recover from backtracks at a force of 7.5 +/- 2 pN, only one-third of the force determined for Escherichia coli RNAP(7,8). We show that backtrack pause durations follow a t(-3/2) power law, implying that during backtracking RNAP II diffuses in discrete base-pair steps, and indicating that backtracks may account for most of RNAP II pauses. Significantly, addition of TFIIS rescued backtracked enzymes and allowed transcription to proceed up to a force of 16.9 +/- 3.4 pN. Taken together, these results describe a regulatory mechanism of transcription elongation in eukaryotes by which transcription factors modify the mechanical performance of RNAP II, allowing it to operate against higher loads.