Molecular Mechanism of Transcription Inhibition by Phage T7 gp2 Protein
Molecular Mechanism of Transcription Inhibition by Phage T7 gp2 Protein
复制标题
DOI：
10.1016/j.jmb.2011.09.029
复制
发表时间：
2011-11-11
影响因子：
5.6
通讯作者：
Severinov, Konstantin
中科院分区：
文献类型：
--
作者：
Mekler, Vladimir;Minakhin, Leonid;Severinov, Konstantin
大肠杆菌T7噬菌体gp 2蛋白是宿主RNA聚合酶（RNAP）的有效抑制剂。gp 2通过结合到β '颚（与下游启动子DNA相互作用的RNAP结构域）来抑制开放启动子复合物的形成。在这里，我们使用了一个工程化的启动子与优化的序列，以获得和表征一个特定的启动子复合物含有RNAP和gp 2。在该复合物中，启动子DNA的局部解链开始，但不传播到包括转录起始点。因此，该复合物在转录上是无活性的。使用高度灵敏的RNAP信标测定，我们进行了定量实时测量RNAP gp 2复合物与启动子DNA和各种启动子片段的特异性结合。以这种方式，剖析了gp 2对RNAP与启动子相互作用的影响。正如预期的那样，gp 2大大降低了RNAP对下游启动子双链体的亲和力。然而，gp 2也抑制RNAP与缺乏与β '颚相互作用的下游启动子DNA的启动子片段的结合。这种抑制是由gp 2介导的RNAP对-10启动子元件下游转录泡的模板和非模板链片段的结合亲和力降低引起的。通过gp 2抑制RNAP与转录泡的单链片段的相互作用是一种新的效应，其可能通过由gp 2结合到β '颚而启动的变构机制发生。(C)2011爱思唯尔有限公司保留所有权利。
Escherichia coli T7 bacteriophage gp2 protein is a potent inhibitor of host RNA polymerase (RNAP). gp2 inhibits formation of open promoter complex by binding to the beta' jaw, an RNAP domain that interacts with downstream promoter DNA. Here, we used an engineered promoter with an optimized sequence to obtain and characterize a specific promoter complex containing RNAP and gp2. In this complex, localized melting of promoter DNA is initiated but does not propagate to include the point of the transcription start. As a result, the complex is transcriptionally inactive. Using a highly sensitive RNAP beacon assay, we performed quantitative real-time measurements of specific binding of the RNAP gp2 complex to promoter DNA and various promoter fragments. In this way, the effect of gp2 on RNAP interaction with promoters was dissected. As expected, gp2 greatly decreased RNAP affinity to downstream promoter duplex. However, gp2 also inhibited RNAP binding to promoter fragments that lacked downstream promoter DNA that interacts with the beta' jaw. The inhibition was caused by gp2-mediated decrease of the RNAP binding affinity to template and non-template strand segments of the transcription bubble downstream of the -10 promoter element. The inhibition of RNAP interactions with single-stranded segments of the transcription bubble by gp2 is a novel effect, which may occur via allosteric mechanism that is set in motion by the gp2 binding to the beta' jaw. (C) 2011 Elsevier Ltd. All rights reserved.