How transcription factors regulate bacterial RNA polymerase activity

该项目将有助于在原子尺度上澄清细菌DNA:RNA转录的机制。一个特别的目的是阐明导致各种转录步骤之间转变的调控过程和这些转变期间贡献蛋白质的构象变化。该项目的长期目标之一是为开发新的抗菌物质奠定基础。在这些研究中，我们将采用光谱、生化和分子生物学的方法。重点研究了RNA聚合酶（RNAP）与Nus因子及相关蛋白在溶液中的相互作用。传统的核磁共振波谱工具箱将被13C和15N特异性和非特异性蛋白质同位素标记方案与渗透氘化相结合而扩大。特别是，通过RNAP亚基的单独表达，单个亚基甲基的13C标记以及完整的多聚体蛋白的重构，RNAP将可用于NMR研究。同样，配体蛋白即使与RNAP配合也可以通过甲基的13C标记进行观察。因此，它将有可能相当准确地定义RNAP和配体蛋白之间的相互作用表面以及描述复合物的几何形状。我们将通过一系列生物化学、分子生物学和体内技术以及分子建模、分子对接和分子动力学计算来补充这些研究。在化学交联实验的同时，蛋白质中单个氨基酸的位点定向变异和蛋白质片段的研究将发挥核心作用。荧光光谱标记或本质荧光蛋白变体以及荧光共振能量转移（FRET）实验将允许更准确地描述转录复合体蛋白质的相互作用。我们将在我们的研究中包括转录因子RfaH，因为RfaH表现出前所未有的折叠行为，即在完整结构域的α -螺旋构象和β -链构象之间的可逆转变。到目前为止，RfaH的独特行为将被研究，特别是我们将尝试识别那些促进这种转变的氨基酸，我们将尝试定义转录过程中启动结构转变的过程，从而激活RfaH。很有可能，特定DNA序列、RNAP和RfaH之间复杂的相互作用是这种转化的关键。

This project will contribute to the clarification of the mechanisms on an atomic scale of bacterial DNA:RNA transcription. A particular aim is the elucidation of the regulatory processes that lead to transitions between the various steps of transcription and the conformational changes of the contributing proteins during these transitions. Among the long term goals of the project is to lay a foundation for the development of new antimicrobial substances. For these studies we will employ spectroscopic, biochemical and molecular biology methods. The focus is the analysis of interactions of RNA polymerase (RNAP) with Nus factors and related proteins by nuclear magnetic resonance (NMR) spectroscopy in solution. The traditional toolbox of NMR spectroscopy will be enlarged by 13C and 15N specific and unspecific protein isotope labeling schemes combined with perdeuteration. In particular, RNAP will be made accessible to NMR studies by separate expression of the RNAP subunits, 13C labeling of methyl groups of the individual subunits, and reconstitution of the intact multimeric protein. Similarly, the ligand proteins will be rendered observable by 13C labeling of their methyl groups even in complex with RNAP. Thus it will be possible to define rather accurately the interaction surfaces between RNAP and ligand proteins as well as describe the geometry of the complexes. We will supplement these studies by a whole range of biochemical, molecular biology, and in vivo techniques as well as by molecular modeling, molecular docking, and molecular dynamics calculations. Site directed variation of individual amino acids of the respective proteins and studies of protein fragments will play a central role, in parallel with chemical cross-linking experiments. Fluorescence spectroscopy of labeled or intrinsically fluorescent protein variants as well as fluorescence resonance energy transfer (FRET) experiments will allow a more accurate description of the mutual interactions of proteins of the transcription complex. We will include in our studies the transcription factor RfaH, as RfaH shows an unprecedented folding behavior, namely the reversible transition between alpha-helical conformation and beta-strand conformation of a complete domain. This so far unique behavior of RfaH will be studied, in particular we will try to identify those amino acids that facilitate this transition, and we will try to define the processes during transcription that initiate the structural changeover and thus the activation of RfaH. Highly likely, intricate interactions between specific DNA sequences, RNAP, and RfaH are key to this transformation.