Structural confirmation of a bent and open model for the initiation complex of T7 RNA polymerase.
Structural confirmation of a bent and open model for the initiation complex of T7 RNA polymerase.
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T7 RNA 聚合酶起始复合物的弯曲和开放模型的结构确认。
DOI：
10.1021/bi061905d
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发表时间：
2007
期刊：
影响因子：
2.9
通讯作者：
Martin,CraigT
中科院分区：
文献类型：
--
作者：
Turingan,RosemaryS;Liu,Cuihua;Hawkins,MaryE;Martin,CraigT
已知 T7 RNA 聚合酶在结合时会诱导其启动子 DNA 弯曲，凝胶位移测定和最近的端到端荧光能量转移距离测量证明了这一点。然而，启动子结合和最初转录复合物的晶体结构缺乏下游 DNA，无法提供有关 DNA 通过蛋白质的整体路径的信息。伸长复合物的晶体结构确实包含下游 DNA，并为初始时完全熔化的气泡结构的模型设计提供了宝贵的指导。在当前的研究中，我们测试了起始复合物的特定结构模型，该模型是通过比对起始和延伸的蛋白质结构的 C 末端区域获得的，然后将下游 DNA 从延伸复合物简单转移到起始复合物上。荧光共振能量转移测量从启动子 DNA 上游点到下游螺旋各点的距离，再现了距离中预期的螺旋周期性，并支持模型的下游 DNA 的方向和定相。该模型还预测了活性位点下游的熔化程度。通过监测 DNA 中不同位置处的荧光碱基类似物，我们绘制了气泡的下游边缘图，从而证实了模型。在没有底物的情况下，最初熔化的气泡包含 7−8 个碱基，并且足以在需要进一步熔化之前合成三碱基转录物。结果表明，尽管蛋白质的 N 端部分和活性位点上游的 DNA 发生了巨大变化，但活性位点下游的 DNA 在起始复合物和延伸复合物中实际上是相同的。
T7 RNA polymerase is known to induce bending of its promoter DNA upon binding, as evidenced by gel-shift assays and by recent end-to-end fluorescence energy transfer distance measurements. Crystal structures of promoter-bound and initially transcribing complexes, however, lack downstream DNA, providing no information on the overall path of the DNA through the protein. Crystal structures of the elongation complex do include downstream DNA and provide valuable guidance in the design of models for the complete melted bubble structure at initiation. In the current study, we test a specific structural model for the initiation complex, obtained by alignment of the C-terminal regions of the protein structures from both initiation and elongation and then simple transferal of the downstream DNA from the elongation complex onto the initiation complex. Fluorescence resonance energy transfer measurement of distances from a point upstream on the promoter DNA to various points along the downstream helix reproduce the expected helical periodicity in the distances and support the model's orientation and phasing of the downstream DNA. The model also makes predictions about the extent of melting downstream of the active site. By monitoring fluorescent base analogues incorporated at various positions in the DNA, we have mapped the downstream edge of the bubble, confirming the model. The initially melted bubble, in the absence of substrate, encompasses 7−8 bases and is sufficient to allow synthesis of a three base transcript before further melting is required. The results demonstrate that despite massive changes in the N-terminal portion of the protein and in the DNA upstream of the active site, the DNA downstream of the active site is virtually identical in both initiation and elongation complexes.