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首页> 外文期刊>Molecular and Cellular Biology >Studies of Nematode TFIIE Function Reveal a Link between Ser-5 Phosphorylation of RNA Polymerase II and the Transition from Transcription Initiation to Elongation
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Studies of Nematode TFIIE Function Reveal a Link between Ser-5 Phosphorylation of RNA Polymerase II and the Transition from Transcription Initiation to Elongation

机译：线虫TFIIE功能的研究揭示了RNA聚合酶II的Ser-5磷酸化与从转录起始到延伸的过渡之间的联系

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The general transcription factor TFIIE plays important roles in transcription initiation and in the transition to elongation. However, little is known about its function during these steps. Here we demonstrate for the first time that TFIIH-mediated phosphorylation of RNA polymerase II (Pol II) is essential for the transition to elongation. This phosphorylation occurs at serine position 5 (Ser-5) of the carboxy-terminal domain (CTD) heptapeptide sequence of the largest subunit of Pol II. In a human in vitro transcription system with a supercoiled template, this process was studied using a human TFIIE (hTFIIE) homolog from Caenorhabditis elegans (ceTFIIEα and ceTFIIEβ). ceTFIIEβ could partially replace hTFIIEβ, whereas ceTFIIEα could not replace hTFIIEα. We present the studies of TFIIE binding to general transcription factors and the effects of subunit substitution on CTD phosphorylation. As a result, ceTFIIEα did not bind tightly to hTFIIEβ, and ceTFIIEβ showed a similar profile for binding to its human counterpart and supported an intermediate level of CTD phosphorylation. Using antibodies against phosphorylated serine at either Ser-2 or Ser-5 of the CTD, we found that ceTFIIEβ induced Ser-5 phosphorylation very little but induced Ser-2 phosphorylation normally, in contrast to wild-type hTFIIE, which induced phosphorylation at both Ser-2 and Ser-5. In transcription transition assays using a linear template, ceTFIIEβ was markedly defective in its ability to support the transition to elongation. These observations provide evidence of TFIIE involvement in the transition and suggest that Ser-5 phosphorylation is essential for Pol II to be in the processive elongation form.
机译：通用转录因子TFIIE在转录起始和向伸长的过渡中起重要作用。但是，在这些步骤中对其功能知之甚少。在这里，我们首次证明TFIIH介导的RNA聚合酶II（Pol II）的磷酸化对于向伸长的转变至关重要。这种磷酸化发生在Pol II最大亚基的羧基末端域（CTD）七肽序列的丝氨酸5位（Ser-5）。在具有超螺旋模板的人体外转录系统中，使用来自秀丽隐杆线虫的人类TFIIE（hTFIIE）同源物（ceTFIIEα和ceTFIIEβ）研究了这一过程。 ceTFIIEβ可以部分替代hTFIIEβ，而ceTFIIEα不能替代hTFIIEα。我们目前的研究TFIIE绑定到一般的转录因子和亚基取代对CTD磷酸化的影响。结果，ceTFIIEα没有与hTFIIEβ紧密结合，并且ceTFIIEβ显示出与人类对应物相似的结合谱，并支持中等水平的CTD磷酸化。使用针对CTD的Ser-2或Ser-5处的磷酸化丝氨酸的抗体，我们发现ceTFIIEβ很少诱导Ser-5磷酸化，但正常情况下诱导Ser-2磷酸化，而野生型hTFIIE则在两者处均诱导磷酸化Ser-2和Ser-5。在使用线性模板的转录过渡测定中，ceTFIIEβ在支持过渡到延伸的能力方面明显缺陷。这些观察结果提供了TFIIE参与过渡的证据，并表明Ser-5磷酸化对Pol II进行性延伸形式至关重要。

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