Exploring RNA polymerase regulation by NMR spectroscopy
Exploring RNA polymerase regulation by NMR spectroscopy
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DOI：
10.1038/srep10825
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发表时间：
2015-06-04
影响因子：
4.6
通讯作者：
Roesch, Paul
中科院分区：
文献类型：
--
作者：
Droegemueller, Johanna;Strauss, Martin;Roesch, Paul
RNA合成是所有生物体的中心过程，RNA聚合酶（RNAP）是其中的关键酶。多亚基RNAP是进化相关的，并受到许多转录因子的严格调控。虽然大肠杆菌RNAP已被广泛研究，只有很少的信息是关于其动力学和瞬态相互作用。然而，这些信息对于完整理解原子细节的转录调控至关重要。为了通过NMR光谱研究RNAP，我们开发了一种高效的程序，用于从单独表达的亚基组装活性RNAP，该亚基允许对单个组分进行特异性标记。我们记录了完整RNAP的异亮氨酸、亮氨酸和缬氨酸甲基的[H-1，C-13]相关光谱以及重组RNAP内单独标记的β '亚基。我们进一步单独产生了所有RNAP亚基，建立了实验来确定某种调节剂与哪个RNAP亚基结合，并鉴定了与NusE结合的β亚基。
RNA synthesis is a central process in all organisms, with RNA polymerase ( RNAP) as the key enzyme. Multisubunit RNAPs are evolutionary related and are tightly regulated by a multitude of transcription factors. Although Escherichia coli RNAP has been studied extensively, only little information is available about its dynamics and transient interactions. This information, however, are crucial for the complete understanding of transcription regulation in atomic detail. To study RNAP by NMR spectroscopy we developed a highly efficient procedure for the assembly of active RNAP from separately expressed subunits that allows specific labeling of the individual constituents. We recorded [H-1, C-13] correlation spectra of isoleucine, leucine, and valine methyl groups of complete RNAP and the separately labeled beta' subunit within reconstituted RNAP. We further produced all RNAP subunits individually, established experiments to determine which RNAP subunit a certain regulator binds to, and identified the beta subunit to bind NusE.