Mapping the conformation of the nucleic acid framework of the T7 RNA polymerase elongation complex in solution using low-energy CD and fluorescence spectroscopy
Mapping the conformation of the nucleic acid framework of the T7 RNA polymerase elongation complex in solution using low-energy CD and fluorescence spectroscopy
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DOI：
10.1016/j.jmb.2006.05.053
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发表时间：
2006-07-21
影响因子：
5.6
通讯作者：
von Hippel, Peter H.
中科院分区：
文献类型：
--
作者：
Datta, Kausiki;Johnson, Neil P.;von Hippel, Peter H.
T7噬菌体延伸复合物是一个极好的模型系统，可用于表征转录的基本步骤。我们通过将预组装的且有RNA引物的DNA“泡状”结构与T7 RNA聚合酶混合，并在启动子处起始转录，形成了有功能的延伸复合物，并且监测了插入在复合物的DNA和RNA支架内特定位置的成对2 - 氨基嘌呤残基的低能圆二色性（CD）和荧光光谱。通过这种方式，当延伸复合物经历核苷酸添加循环的各个步骤时，我们能够探测这些位置的碱基和碱基对局部构象的特定变化。使用插入在特定核酸位置的成对2 - 氨基嘌呤残基作为探针的优势在于，在波长>300 nm时，复合物的其余部分在光谱上是“透明的”。因此，通过结合圆二色性和荧光测量，我们获得了在功能复合物内每个位置独特适用的结构和动态信息。通过这种方式，我们绘制了转录核心步骤的细节图，包括转录泡的形成和移位、RNA - DNA杂合体的形成和解旋、新生RNA通过聚合酶出口通道的过程，以及控制转录保真度的模板控制的NTP选择过程中的事件。这种方法确定了生理条件下延伸过程的特定结构方面，并且可以扩展用于研究转录调控的其他关键方面，例如终止、编辑、暂停等，这些方面涉及转录复合物的核酸框架内的构象重排。（c）2006爱思唯尔有限公司。保留所有权利。
The bacteriophage T7 elongation complex is an excellent model system in which to characterize the fundamental steps of transcription. We have formed functional elongation complexes, by mixing preassembled and RNA-primed DNA "bubble" constructs with T7 RNA polymerase and by initiating transcription at promoters, and have monitored the low-energy CD and fluorescence spectra of pairs of 2-aminopurine residues that have been inserted at defined sites within the DNA and RNA scaffold of the complex. In this way, we have been able to probe specific changes in the local conformations of the bases and base-pairs at these positions as the elongation complex goes through the various steps of the nucleotide addition cycle. The advantage of using pairs of 2-aminopurine residues, inserted at defined nucleic acid positions, as probes, is that the rest of the complex is spectrally "transparent" at wavelengths > 300 nm. Thus, by combining CD and fluorescence measurements we obtain both structural and dynamic information that applies uniquely at each position within the functioning complex. In this way, we have mapped the details of steps central to transcription, including the formation and translocation of the transcription bubble, the formation and unwinding of the RNA-DNA hybrid, the passage of the nascent RNA through the exit channel of the polymerase, and the events of the template-controlled NTP selection process that controls transcriptional fidelity. This approach defines specific structural aspects of the elongation process under physiological conditions, and can be extended to examine other key aspects of transcriptional regulation, such as termination, editing, pausing, etc., that involve conformational rearrangements within the nucleic acid framework of the transcription complex. (c) 2006 Elsevier Ltd. All rights reserved.