Double-stranded DNA translocase activity of transcription factor TFIIH and the mechanism of RNA polymerase II open complex formation
Double-stranded DNA translocase activity of transcription factor TFIIH and the mechanism of RNA polymerase II open complex formation
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DOI：
10.1073/pnas.1417709112
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发表时间：
2015-03-31
影响因子：
11.1
通讯作者：
Hahn, Steven
中科院分区：
文献类型：
--
作者：
Fishburn, James;Tomko, Eric;Hahn, Steven
RNA 聚合酶 II (Pol II) 开放复合物 (OC) 的形成需要由转录因子 TFIIH 解旋酶相关亚基 XPB/Ssl2 介导的 DNA 解旋。由于 XPB/Ssl2 在 DNA 解旋位置下游结合 DNA，因此它无法使用传统的解旋酶机制发挥作用。在这里，我们证明酵母 TFIIH 含有 Ssl2 依赖性双链 DNA 转位酶活性。 Ssl2 沿着 5' 端的一条 DNA 链进行追踪。 3' 方向，意味着它使用非模板启动子链将下游 DNA 卷入 Pol II 裂口，产生扭转应变并导致 DNA 解旋。对 Ssl2 和 TFIIH 的 DNA 依赖性 ATP 酶活性的分析表明，Ssl2 具有大约一圈 DNA 的持续合成能力，与转录起始过程中解开的 DNA 长度一致。我们的结果可以解释为什么维持 OC 需要持续的 ATP 水解以及 TFIIH 在启动子逃逸中的功能。我们的结果还表明 XPB/Ssl2 在 DNA 修复过程中使用这种易位酶机制，而不是物理楔入打开受损的 DNA。
Formation of the RNA polymerase II (Pol II) open complex (OC) requires DNA unwinding mediated by the transcription factor TFIIH helicase-related subunit XPB/Ssl2. Because XPB/Ssl2 binds DNA downstream from the location of DNA unwinding, it cannot function using a conventional helicase mechanism. Here we show that yeast TFIIH contains an Ssl2-dependent double-stranded DNA translocase activity. Ssl2 tracks along one DNA strand in the 5'. 3' direction, implying it uses the nontemplate promoter strand to reel downstream DNA into the Pol II cleft, creating torsional strain and leading to DNA unwinding. Analysis of the Ssl2 and DNA-dependent ATPase activity of TFIIH suggests that Ssl2 has a processivity of approximately one DNA turn, consistent with the length of DNA unwound during transcription initiation. Our results can explain why maintaining the OC requires continuous ATP hydrolysis and the function of TFIIH in promoter escape. Our results also suggest that XPB/Ssl2 uses this translocase mechanism during DNA repair rather than physically wedging open damaged DNA.