Structure and Dynamics of AsiA and the AsiA-sigma70 Complex

描述（由申请人提供）：AsiA蛋白是一种新型转录调节因子，是T4噬菌体asiA基因的产物，对细菌细胞具有致死性。这种蛋白质的天然抗生素倾向，结合它与细胞中高度保守的机制元件-RNA聚合酶复合物-对基因转录和基因转录调控至关重要的事实，表明可以基于AsiA及其功能机制开发新型广谱抗生素。由于病原菌对现有抗生素的耐药性不断增加，新的抗生素和抗生素开发策略对于对抗细菌感染和维护公共健康至关重要。 我们研究的长期目标是了解转录调控的机制和结构细节。我们目前的重点是在细菌的转录起始和分子间的识别和相互作用的事件，调节启动过程。因此，AsiA蛋白具有重大意义。AsiA是一个独特的分子开关，在宿主- E转录噬菌体基因组期间决定启动子选择。coli-RNA聚合酶，是控制T4基因组转录方向和进程的主要因素之一。AsiA与RNA聚合酶全酶的σ 70亚基紧密结合。这种紧密的相互作用被认为是其功能的全部原因。然而，我们最近发现，AsiA也结合到RNA聚合酶核心的其他成分，似乎也通过螺旋-转角-螺旋DNA结合基序识别DNA。这些属性形成了我们的总体假设的基础，即AsiA及其直系同源物是独特的分子适配器，其通过与sigma 70、β亚基以及在某些情况下还与启动子DNA的相互作用介导转录起始复合物的结构重组以及启动子识别和选择。我们的目标是（1）检查AsiA-beta亚基相互作用的结构和功能后果，（2）定义AsiA相关蛋白家族的转录调节机制，（3）确定AsiA-DNA相互作用如何促进AsiA的转录调节。我们将使用NMR建立蛋白质和蛋白质复合物的高分辨率结构模型，将联合收割机突变分析与表面等离子体共振相结合，以确定单个氨基酸对复合物形成的亲和力和自由能贡献，并使用化学交联和体外转录方法来表征与DNA的相互作用。总的来说，我们的研究结果将提供AsiA和相关蛋白质转录调控的复杂结构和功能解释，将为蛋白质结构和蛋白质相互作用领域提供新的见解，并将提供追求药物设计所需的关键化学观点，以模仿AsiA的抗生素特性。

DESCRIPTION (provided by applicant): The AsiA protein, a novel transcriptional regulator and product of the T4-bacteriophage asiA gene, is lethal to bacterial cells. The natural antibiotic tendencies of this protein, combined with the fact that it interacts with highly conserved elements of the machinery in cells - the RNA polymerase complex - central to gene transcription and regulation of gene transcription, suggests that novel, broad-spectrum antibiotics can be developed based on AsiA and the mechanism by which it functions. Because of the increasing resistance of pathogenic bacteria to available antibiotics, new antibiotics and strategies for antibiotic development are critical for fighting bacterial infections and maintaining the public health. The long-term objective of our research is to understand the mechanistic and structural details of transcription regulation. Our immediate focus is on transcription initiation in bacteria and intermolecular recognition and interaction events that regulate the initiation process. Accordingly, the AsiA protein is of significant interest. AsiA is a unique molecular switch that dictates promoter selection during transcription of the phage genome by the host - E. coli - RNA polymerase and is one of the principal factors governing the direction and progression of transcription of the T4 genome. AsiA binds tightly to the sigma70 subunit of the RNA polymerase holoenzyme. This tight interaction was thought to be wholly responsible for its function. However, we have recently discovered that AsiA also binds to other components of the RNA polymerase core and appears also to recognize DNA via a helix-turn-helix DNA binding motif. These attributes form the foundation of our overall hypothesis that AsiA and its orthologs are unique molecular adapters that mediate the structural reorganization of the transcription initiation complex and promoter recognition and selection via interactions with sigma70, the beta subunit, and, under some circumstances, also the promoter DNA. Our aims are (1) to examine the structural and functional consequences of the AsiA-beta subunit interactions, (2) to define the mechanism of transcription regulation by a family of proteins related to AsiA, and (3) to determine how AsiA-DNA interactions contribute to transcription regulation by AsiA. We will build high resolution structural models of the proteins and protein complexes using NMR, combine mutational analyses with surface plasmon resonance to determine affinities and free energy contributions of individual amino acids to complex formation, and use chemical cross-linking and in vitro transcription methods to characterize interactions with DNA. Overall, our results will provide an intricate structural and functional account of transcription regulation by AsiA and related proteins, will provide new insight into the realms of protein structure and protein interactions in general, and will provide the critical chemical view necessary for pursuit of drug design to mimic the antibiotic properties of AsiA.