Functional architecture of T7 RNA polymerase transcription complexes
Functional architecture of T7 RNA polymerase transcription complexes
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DOI：
10.1016/j.jmb.2007.05.070
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发表时间：
2007-08-10
影响因子：
5.6
通讯作者：
Sousa, Rui
中科院分区：
文献类型：
--
作者：
Nayak, Dhananjaya;Guo, Qing;Sousa, Rui
噬菌体 T7 RNA 聚合酶是广泛分布的单亚基 RNA 聚合酶家族中最具特征的成员。 T7 RNA聚合酶起始和延伸复合物的晶体结构提供了关于RNA聚合酶与启动子和转录泡相互作用的大量详细信息，但是起始复合物结构中模板熔化区域下游不存在DNA，并且延伸复合物结构中转录泡上游不存在DNA，这意味着我们对T7 RNA聚合酶转录复合物功能结构的了解仍然不完整。在这里，我们使用位点特异性束缚的化学核酸酶和定向 T7 RNAP 突变体的功能表征来揭示 T7 RNAP 起始和延伸复合物中转录泡两侧的双链体 DNA 的结构，并定义这些双链体元件相互作用的功能。我们发现，在延伸和起始过程中都存在与赖氨酸残基簇 (K711/K713/K714) 形成的下游双链体相互作用，它们有助于稳定下游 DNA 中的弯曲，这对于启动子打开非常重要。还发现延伸复合物中的上游 DNA 在转录泡的上游边缘急剧弯曲，从而形成上游双链体：聚合酶相互作用，从而有助于延伸复合物的稳定性。 (C) 2007 Elsevier Ltd. 保留所有权利。
Bacteriophage T7 RNA polymerase is the best-characterized member of a widespread family of single-subunit RNA polymerases. Crystal structures of T7 RNA polymerase initiation and elongation complexes have provided a wealth of detailed information on RNA polymerase interactions with the promoter and transcription bubble, but the absence of DNA downstream of the melted region of the template in the initiation complex structure, and the absence of DNA upstream of the transcription bubble in the elongation complex structure means that our picture of the functional architecture of T7 RNA polymerase transcription complexes remains incomplete. Here, we use the site-specifically tethered chemical nucleases and functional characterization of directed T7 RNAP mutants to both reveal the architecture of the duplex DNA that flanks the transcription bubble in the T7 RNAP initiation and elongation complexes, and to define the function of the interactions made by these duplex elements. We find that downstream duplex interactions made with a cluster of lysine residues (K711/K713/ K714) are present during both elongation and initiation, where they contribute to stabilizing a bend in the downstream DNA that is important for promoter opening. The upstream DNA in the elongation complex is also found to be sharply bent at the upstream edge of the transcription bubble, thereby allowing formation of upstream duplex:polymerase interactions that contribute to elongation complex stability. (C) 2007 Elsevier Ltd. All rights reserved.