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首页> 外文期刊>Biochemistry >The T7 RNA polymerase intercalating hairpinis importat for promoter opening during initiation but
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The T7 RNA polymerase intercalating hairpinis importat for promoter opening during initiation but

机译：T7 RNA聚合酶插入发夹对于启动过程中启动子的打开很重要，但

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The recently described crystal structures of a T7RNAP—promoter complex and an initial transcription complex reveal a /3-hairpin which inserts between the template and nontemplate strands of the promoter [Cheetham, G. M., et al. (1999) Nature 399, 80; Cheetham, G. M., et al. (1999) Science 286, 2305]. A stacking interaction between the exposed DNA bases and a valine at the tip of this hairpin may be especially important for stabilizing the opened promoter during initiation. It has been suggested that this hairpin may also be important for holding the transcription bubble open during transcript elongation, and a proposed model for how the RNA exits the transcription complex implies that this hairpin may also help displace the RNA from the template strand. To test these hypotheses, we have characterized both point and deletion mutants of this element. We find that these mutants exhibit reduced activity on linear, double-stranded templates but not on supercoiled or partially single-stranded templates. Probing of promoter—polymerase complexes, initial transcription complexes, and elongation complexes with KIVInO4 and a single-strand specific endonuclease reveals that the mutants have greatly reduced promoter unwinding activity during initiation. However, the structure and stability of the transcription bubble during elongation are not altered in the mutant enzymes, and RNA displacement activity is also normal. Thus, the T7RNAP intercalating hairpin is important, though not essential, for stabilizing the opened promoter during initiation, but is not important for RNA displacement or for transcription bubble structure or stability during elongation.
机译：最近描述的T7RNAP-启动子复合物和初始转录复合物的晶体结构揭示了插入在启动子的模板链和非模板链之间的/​​ 3-发夹[Cheetham，G.M。，等。 （1999）Nature 399，80; Cheetham，G.M。等。 （1999）Science 286，2305]。在发夹末端，暴露的DNA碱基与缬氨酸之间的堆积相互作用对于在启动过程中稳定打开的启动子可能尤其重要。已经提出，该发夹对于在转录物延伸期间保持转录气泡开放也可能是重要的，并且所提出的RNA如何离开转录复合物的模型暗示了该发夹还可以帮助从模板链上置换RNA。为了检验这些假设，我们已经对该元件的点突变和缺失突变进行了表征。我们发现这些突变体在线性，双链模板上表现出降低的活性，但在超螺旋或部分单链模板上却没有。对启动子-聚合酶复合物，初始转录复合物和与KIVInO4的延伸复合物以及单链特异性核酸内切酶的探测表明，突变体在启动过程中大大降低了启动子的解链活性。但是，在突变酶中，伸长过程中转录气泡的结构和稳定性没有改变，并且RNA置换活性也是正常的。因此，尽管不是必需的，T7RNAP插入发夹对于启动过程中稳定打开的启动子很重要，但对于RNA置换或转录气泡结构或延伸过程中的稳定性而言并不重要。

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