How transcription factors regulate bacterial RNA polymerase activity

该项目将有助于澄清细菌DNA：RNA转录的原子尺度机制。一个特别的目的是阐明的监管过程，导致转录的各个步骤之间的转换和这些转换过程中的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。对于这些研究，我们将采用光谱，生物化学和分子生物学方法。重点是通过核磁共振（NMR）光谱分析RNA聚合酶（RNAP）与Nus因子和相关蛋白质的相互作用。传统的核磁共振波谱工具箱将通过13 C和15 N特异性和非特异性蛋白质同位素标记方案与全氘化相结合来扩大。特别是，RNAP将通过RNAP亚基的单独表达、单个亚基的甲基的13 C标记和完整的多聚体蛋白的重建而可用于NMR研究。类似地，配体蛋白质将通过其甲基的13 C标记而变得可观察，即使在与RNAP复合的情况下。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，以及描述的几何形状的复合物。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。与化学交联实验平行，各个蛋白质的单个氨基酸的定点变异和蛋白质片段的研究将发挥核心作用。标记的或固有荧光蛋白变体的荧光光谱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物的蛋白质的相互作用。我们将在我们的研究中包括转录因子RfaH，因为RfaH显示出前所未有的折叠行为，即完整结构域的α-螺旋构象和β-链构象之间的可逆转变。我们将研究RfaH迄今为止的独特行为，特别是我们将尝试识别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换的过程，从而激活RfaH。极有可能的是，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.