The T7 RNA polymerase intercalating hairpin is important for promoter opening during initiation but not for RNA displacement or transcription bubble stability during elongation
The T7 RNA polymerase intercalating hairpin is important for promoter opening during initiation but not for RNA displacement or transcription bubble stability during elongation
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DOI：
10.1021/bi002716c
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发表时间：
2001-04-03
期刊：
影响因子：
2.9
通讯作者：
Sousa, R
中科院分区：
文献类型：
--
作者：
Brieba, LG;Sousa, R
最近描述的T7 RNAP-启动子复合物和初始转录复合物的晶体结构揭示了插入启动子的模板和非模板链之间的P-发夹[Cheetham，G. M.，等人（1999）Nature 399，80; Cheetham，G. M.，等人（1999）Science 286，2305]。暴露的DNA碱基和该发夹顶端的缬氨酸之间的堆积相互作用对于在起始期间稳定打开的启动子可能是特别重要的。有人提出，这种发夹可能也是重要的保持转录泡打开在转录延长，和一个提出的模型，RNA如何退出转录复合物意味着，这种发夹也可能有助于取代RNA从模板链。为了验证这些假设，我们的特点是点和缺失突变体的这个元素。我们发现，这些突变体表现出线性，双链模板，但不是超螺旋或部分单链模板的活动减少。探测的启动子聚合酶复合物，初始转录复合物，并延长复合物与高锰酸钾和单链特异性核酸内切酶揭示，突变体在启动过程中大大降低了启动子解旋活性。然而，在延伸过程中转录泡的结构和稳定性在突变酶中没有改变，并且RNA置换活性也是正常的。因此，T7 RNAP插入发夹对于在起始期间稳定打开的启动子是重要的，尽管不是必需的，但对于RNA置换或对于转录泡结构或延伸期间的稳定性是不重要的。
The recently described crystal structures of a T7RNAP-promoter complex and an initial transcription complex reveal a P-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 KMnO4 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.