STRUCTURE AND DYNAMICS OF ASIA AND THE ASIA 670 COMPLEX

asiA基因噬菌体T4的AsiA蛋白产物的存在的抗生效果是由于其与细菌σ因子的紧密相互作用，其实例是其与E.杆菌 该研究的总体目标是提供一个结构和动力学平台，以了解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。 除了内在相关的，以促进我们的portein-蛋白质相互作用的fundamentsls一般的理解，结果是一个必要的前奏，设计一个新的广谱利用AsiA模拟。AsiA蛋白与E.大肠杆菌RNA聚合酶显然是控制T4基因组转录方向和进程的主要因素之一。 此外，由于AsiA的过表达对细菌的致死作用，AsiA-sigma 70界面可以代表一类令人兴奋的新型广谱抗生素及其靶标的原型。 这项研究将研究AsiA在溶液中的结构和动力学，以及与E.大肠杆菌RNA聚合酶，主要使用核磁共振（NMR）技术。 AsiA蛋白及其与Sigma 70的AsiA结合结构域的复合物的高分辨率结构模型将使用距离几何/模拟退火方法和NMR衍生的约束来生成。 氢交换和15 N和13 C相关技术将用于确定AsiA和AsiA sigma 70复合体的主链和侧链的运动分布、幅度和时间尺度。 结合研究和新的高压NMR研究将揭示AsiA-sigma 70相互作用的位点特异性热力学，将提供对一般情况下静态和动态事件相互作用的复杂解释，并将为追求基于结构的药物设计以模拟AsiA的抗生素性质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.