Structure and Dynamics of AsiA and the AsiA-sigma70 Complex

AsiA 和 AsiA-sigma70 复合体的结构和动力学

• 批准号: 8000018
• 负责人: JEFFREY L URBAUER
• 金额: $ 12.76万
• 依托单位: UNIVERSITY OF GEORGIA
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-01-07 至 2011-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8000018
• 关键词: Accounting; Affinity; Amino Acid Sequence; Amino Acids; Antibiotics; Asia; Bacteria; Bacterial Infections; Bacteriophage T4; Bacteriophages; Binding; Cells; Chemicals; Complex; DNA; DNA Binding; DNA-Directed RNA Polymerase; Development; Dissociation; Distant; Drug Design; Elements; Escherichia coli; Event; Family; Family member; Foundations; Free Energy; Genes; Genetic Transcription; Genome; Goals; Helix-Turn-Helix Motifs; Holoenzymes; In Vitro; Indium; Individual; Mediating; Methods; Molecular; Orthologous Gene; Play; Polymerase; Process; Property; Protein Family; Proteins; Public Health; Regulation; Relative (related person); Research; Research Personnel; Resistance; Resolution; Role; Sigma Factor; Solutions; Structural Models; Structure; Surface Plasmon Resonance; Surgical Flaps; Transcription Initiation; Transcriptional Regulation; base; crosslink; fighting; insight; interest; member; novel; pathogenic bacteria; programs; promoter; protein complex; protein structure

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.

描述（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，加上它与细胞机器中高度保守的元件（RNA聚合酶复合物）相互作用这一事实，对于基因转录和基因转录的调节至关重要，这表明可以基于AsiA及其作用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强，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众健康至关重要。 我们研究的长期目标是了解转录调控的机制和结构细节。我们当前的重点是细菌中的转录起始以及调节起始过程的分子间识别和相互作用事件。因此，AsiA 蛋白引起了人们的极大兴趣。 AsiA 是一种独特的分子开关，在宿主 - 大肠杆菌 - RNA 聚合酶转录噬菌体基因组期间决定启动子选择，并且是控制 T4 基因组转录方向和进展的主要因素之一。 AsiA 与 RNA 聚合酶全酶的 sigma70 亚基紧密结合。人们认为这种紧密的相互作用是其功能的全部原因。然而，我们最近发现 AsiA 也与 RNA 聚合酶核心的其他成分结合，并且似乎还通过螺旋-转角-螺旋 DNA 结合基序识别 DNA。这些属性构成了我们总体假设的基础，即 AsiA 及其直系同源物是独特的分子接头，通过与 sigma70、β 亚基以及在某些情况下还与启动子 DNA 相互作用来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-β亚基相互作用的结构和功能后果，（2）定义与AsiA相关的蛋白质家族的转录调节机制，以及（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，将突变分析与表面等离子共振相结合，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，我们的结果将为AsiA和相关蛋白的转录调控提供复杂的结构和功能解释，将为蛋白质结构和蛋白质相互作用领域提供新的见解，并将为追求模拟AsiA抗生素特性的药物设计提供必要的关键化学观点。