STRUCTURE AND DYNAMICS OF ASIA AND THE ASIA 670 COMPLEX

亚洲基因噬菌体T4的亚洲蛋白产物的存在对抗生素的影响是由于它与细菌Sigma因子的紧密相互作用，例如它与E.Coli的Sigma 70的整合。这项研究的总体目标是提供一个结构和动态的平台，以便在不同的分辨率下了解亚洲和细菌Sigma因子之间的相互作用，从而阐明亚洲对细菌转录的调控机制。除了在本质上与进一步了解蛋白-蛋白质相互作用的基本原理有关外，这些结果也是设计一种新型广谱抗菌药物开发亚洲仿制药的必要前奏。ASIA蛋白与E.Coli RNA聚合酶Sigma 70亚基的结合显然是控制T4基因组转录方向和进程的主要因素之一。此外，由于ASIA的过度表达对细菌的致死作用，ASIA-Sigma 70界面可能代表一类令人兴奋的新型硼系抗生素的原型和它的靶标。这项拟议的研究将主要利用核磁共振技术，研究亚洲在溶液中的结构和动力学，以及与大肠杆菌RNA聚合酶的sigma 70亚单位的亚洲结合域的复合体。利用距离几何/模拟退火法和核磁共振推导的约束条件，将建立亚洲蛋白及其与Sigma 70的亚洲结合结构域的高分辨率结构模型。将使用氢交换和15N和13C关联技术来确定亚洲和亚洲西格玛70复合体的主链和侧链的运动的分布、大小和时间尺度。结合研究和新颖的高压核磁共振研究将揭示ASIA-sigma70相互作用的特定部位的热力学，将提供对GERNAL中静态和动态事件相互作用的复杂描述，并将为寻求基于结构的药物设计以模拟亚洲的抗生素特性提供必要的关键化学观点。

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.