STRUCTURE AND DYNAMICS OF ASIA AND THE ASIA 670 COMPLEX

亚洲基因噬菌体T4的亚洲蛋白产物存在的抗生素后果是由于它与细菌的sigma因子紧密相互作用，其中一个例子是它与大肠杆菌的sigma 70的结合。本研究的总体目标是提供一个结构和动态的平台，在原子分辨率上理解AsiA和细菌sigma因子之间的相互作用，从而阐明AsiA调控细菌转录的机制。除了本质上与我们进一步了解蛋白质蛋白相互作用的基本原理有关外，这些结果是设计一种利用亚洲拟态的新型广谱抗生素的必要前奏。AsiA蛋白与大肠杆菌RNA聚合酶的sigma 70亚基的结合显然是控制T4基因组转录方向和进展的主要因素之一。此外，由于过表达AsiA蛋白对细菌的杀伤作用，AsiA-sigma 70界面可能代表了一类令人兴奋的新型单谱抗生素及其靶点的原型。该研究将主要使用核磁共振技术来检测单独溶液中AsiA的结构和动力学，以及与大肠杆菌RNA聚合酶sigma 70亚基AsiA结合区域的复配。将使用距离几何/模拟退火方法和核磁共振衍生约束生成AsiA蛋白及其具有sigma 70 AsiA结合域的复合物的高分辨率结构模型。氢交换和15N和13C弛化技术将用于确定AsiA和AsiA sigma 70配合物的主链和侧链的分布、大小和运动的时间尺度。结合研究和新型高压核磁共振研究将揭示AsiA-sigma70相互作用的位点特异性热力学，将提供静态和动态事件相互作用的复杂描述，并将为追求基于结构的药物设计提供必要的关键化学观点，以模仿AsiA的抗生素特性。

The antibiotic consequences of the presence of the AsiA protein product of the asiA gene bacgteriophage T4, are due to its tight interaction with bacteral sigma factors, an example of which is its intaration with sigma 70 of E. Coli. The overall goal of the proposed research is to provide a structural and dynamic platform for understanding, at atamoic resolution, the interation between AsiA and bacterial sigma factors, and as result, to elucidate the mechanism of regulation of bacterial transcription by AsiA. Besides being intrinically relevant to furthering our understanding of the fundamentsls of portein-protein interactions in general, the results are an obligatory prelude to the design of a novel broad spectrumantibioic exploiting AsiA mimicry. The binding of the AsiA protein to the sigma 70 subunit of the E. Coli RNA polymerase is clearly one of the principle factors governing the direction and progression of transcription of the T4 genome. In addition, because of lethal effects on bacteria of overexpression of AsiA, and AsiA-sigma 70 interface could represent the prototype for an exciting novel class of borad spectrum antibiotics and it's target. The research proposed will examine the structure and dynamics of AsiA in solution alone and ocmplexed to the AsiA binding domain of the sigma 70 subunit of the e. Coli RNA polymerase, using primarily nuclear magnetic resonance (NMR) techniques. High resolution structural models fo rthe AsiA protein and its complex with the AsiA binding domain of sigma 70 will be generated using distance geometry/simulated annealing methods and NMR-derieved restraints. Hydrogen exchange and 15N and 13C relatxation techniques will be used to determine the distribution, magnitude, and timescale of motions of the backbone and side chains of AsiA and the AsiA sigma 70 complex. Binding studies and novel high pressure NMR studies wil reveal the site-specific thermodynamics of the AsiA-sigma70 interaction, will provide an intricate account of the static and dynamic events interactions in gerneral, and will provide the critical chemical view necessary for pursuit of structure-based drug design to mimic the antibiotic properties of AsiA.