Determination of RNA polymerase binding surfaces of transcription factors by NMR spectroscopy
Determination of RNA polymerase binding surfaces of transcription factors by NMR spectroscopy
复制标题
DOI：
10.1038/srep16428
复制
发表时间：
2015-11-12
影响因子：
4.6
通讯作者：
Knauer, Stefan H.
中科院分区：
文献类型：
--
作者：
Droegemueller, Johanna;Strauss, Martin;Knauer, Stefan H.
在细菌中，RNA聚合酶(RNAP)是转录的中心酶，受N-利用物质(NUS)转录因子的调控。这些因素中有几个与RNAP直接相互作用，并且只是暂时的，以调节其功能。由于这些相互作用的细节在很大程度上尚不清楚，我们通过核磁共振(NMR)光谱探索了大肠杆菌(E.Coli)NUS因子的RNAP结合面。用质子化RNAP滴定具有[H-1，C-13]标记的Val、Leu和Ile残基的全氚因子。在与NusG和RNAP的N-末端结构域(NTD)验证后，我们确定了NUSE的RNAP结合位点。它与NusG C-末端结构域的NUSE相互作用面重叠，表明RNAP和NusG竞争NUSE，并暗示了NUSE：RNAP相互作用的可能作用，例如在抗终止和直接转录：翻译偶联中。我们用核磁共振波谱解析了NusA-NTD的溶液结构，用与NusG-NTD相同的方法确定了它的RNAP结合部位，并在这里提出了NusA-NTD：RNAP：RNA络合物的详细模型。
In bacteria, RNA polymerase (RNAP), the central enzyme of transcription, is regulated by N-utilization substance (Nus) transcription factors. Several of these factors interact directly, and only transiently, with RNAP to modulate its function. As details of these interactions are largely unknown, we probed the RNAP binding surfaces of Escherichia coli (E. coli) Nus factors by nuclear magnetic resonance (NMR) spectroscopy. Perdeuterated factors with [H-1, C-13]-labeled methyl groups of Val, Leu, and Ile residues were titrated with protonated RNAP. After verification of this approach with the N-terminal domain (NTD) of NusG and RNAP we determined the RNAP binding site of NusE. It overlaps with the NusE interaction surface for the NusG C-terminal domain, indicating that RNAP and NusG compete for NusE and suggesting possible roles for the NusE: RNAP interaction, e.g. in antitermination and direct transcription: translation coupling. We solved the solution structure of NusA-NTD by NMR spectroscopy, identified its RNAP binding site with the same approach we used for NusG-NTD, and here present a detailed model of the NusA-NTD: RNAP: RNA complex.