TFIIS enhances transcriptional elongation through an artificial arrest site in vivo
TFIIS enhances transcriptional elongation through an artificial arrest site in vivo
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DOI：
10.1128/mcb.21.13.4162-4168.2001
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发表时间：
2001-07-01
影响因子：
5.3
通讯作者：
Struhl, K
中科院分区：
文献类型：
--
作者：
Kulish, D;Struhl, K
RNA聚合酶II的转录延伸已经在体外得到了很好的研究，但由于缺乏直接和特异性的测定，对体内这一过程的理解受到限制。在这里，我们设计了一个特定的测定转录延长在体内，涉及一个人工逮捕（ARTAR）网站设计的热力学理论的DNA依赖的转录逮捕在体外。转录分析和染色质免疫沉淀实验表明，ARTAR位点可以在体内远离启动子的位置处阻止Pol II。TFIIS可以抵消这种逮捕，从而证明它具有体内转录antiarrest活性。出乎意料的是，ARTAR位点在高转录激活条件下不起作用，除非细胞暴露于推测影响体内延伸的条件（6-氮尿嘧啶或降低的温度）。相反，TFIIS在高转录激活条件下影响基因表达，但不影响低转录激活条件。我们的研究结果提供了物理证据的不连续性在体内的转录延伸，他们认为，转录阻滞位点和TFIIS的功能的重要性是强烈的转录激活水平的影响。
Transcriptional elongation by RNA polymerase II has been well studied in vitro, but understanding of this process in vivo has been limited by the lack of a direct and specific assay. Here, we designed a specific assay for transcriptional elongation in vivo that involves an artificial arrest (ARTAR) site designed from a thermodynamic theory of DNA-dependent transcriptional arrest in vitro. Transcriptional analysis and chromatin immunoprecipitation experiments indicate that the ARTAR site can arrest Pol II in vivo at a position far from the promoter. TFIIS can counteract this arrest, thereby demonstrating that it possesses transcriptional antiarrest activity in vivo. Unexpectedly, the ARTAR site does not function under conditions of high transcriptional activation unless cells are exposed to conditions (6-azauracil or reduced temperature) that are presumed to affect elongation in vivo. Conversely, TFIIS affects gene expression under conditions of high, but not low, transcriptional activation. Our results provide physical evidence for the discontinuity of transcription elongation in vivo, and they suggest that the functional importance of transcriptional arrest sites and TFIIS is strongly influenced by the level of transcriptional activation.