Eukaryotic mRNA transcription initiation is directed by the formation of the megadalton-sized preinitiation complex (PIC). After PIC formation, double-stranded DNA (dsDNA) is unwound to form a single-stranded DNA bubble, and the template strand is loaded into the polymerase active site. DNA opening is catalyzed by Ssl2 (XPB), the dsDNA translocase subunit of the basal transcription factor TFIIH. In yeast, transcription initiation proceeds through a scanning phase during which downstream DNA is searched for optimal start sites. Here, to test models for initial DNA opening and start-site scanning, we measure the DNA-bubble sizes generated by Saccharomyces cerevisiae PICs in real time using single-molecule magnetic tweezers. We show that ATP hydrolysis by Ssl2 opens a 6-base-pair (bp) bubble that grows to 13 bp in the presence of NTPs. These observations support a two-step model wherein ATP-dependent Ssl2 translocation leads to a 6-bp open complex that RNA polymerase II expands via NTP-dependent RNA transcription.

中文翻译：

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TFIIH在RNAP II转录起始和起始位点扫描期间生成六碱基对的开放复合体

真核mRNA转录起始是由兆达尔顿大小的预起始复合物（PIC）的形成指导的。PIC形成后，解开双链DNA（dsDNA）以形成单链DNA气泡，并将模板链加载到聚合酶活性位点中。DNA的开放由基础转录因子TFIIH的dsDNA translocase亚基Ssl2（XPB）催化。在酵母中，转录起始通过扫描阶段进行，在该扫描阶段中搜索下游DNA以寻找最佳起始位点。在这里，为了测试用于初始DNA开放和起始位点扫描的模型，我们测量了酿酒酵母产生的DNA气泡大小使用单分子磁性镊子实时PIC。我们显示Ssl2的ATP水解打开了一个6个碱基对（bp）的气泡，该气泡在NTP的存在下增长到13 bp。这些观察结果支持两步模型，其中ATP依赖的Ssl2易位导致6 bp的开放复合物，RNA聚合酶II通过NTP依赖的RNA转录进行扩增。