Function and Metabolism of RNA

描述（由申请人提供）：通用酶核糖核酸酶P（RNase P）通过切割转移RNA（tRNA）进行转移RNA（tRNA）的成熟。RNase P是一种不寻常的酶，因为它是一种核糖核蛋白（RNP），其催化组分由RNA而不是通常的蛋白质组成。RNA-RNA和RNA-蛋白质相互作用对细胞活动至关重要，因此RNA酶P提供了分子结构和功能的具体例子，对其他细胞功能具有更广泛的影响。该提案的总体目标是确定细菌RNase P RNA（~400个核苷酸）及其与RNase P蛋白（~120个氨基酸）和tRNA底物（~80 nt）的复合物的结构。了解这些结构对于RNase P研究的进展至关重要。该实验室长期以来一直致力于这一目标，取得了相当大的成功，首先与生化方法，最近与确定的晶体结构的RNase P RNA从嗜热脂肪芽孢杆菌在3.3？设计了一种在3.6 ℃下快速结晶的RNA。然而，部分晶体结构被扭曲或未解析，全酶和与tRNA的三元复合物的具体结构尚不清楚。即使是活动地点的位置也是值得怀疑的。在目前的解析阶段，机械相关的属性，如金属，氢键网络和沃茨没有辨别。此外，对溶液结构知之甚少，这是一个重要的考虑因素，因为任何晶体结构中都存在潜在的扭曲和无序。为了解决这些和其他问题，我们建议继续进行晶体学研究，以确定与RNase P相关的分子结构。为了补充、测试和完善结构模型，小角X射线散射（SAXS）被用来确定溶液结构以进行比较。SAXS结果与可用的晶体结构信息相结合，将允许将全局溶液包络建模为~10？，晶体学方面的努力包括对最近数据集的持续分析，以及筛选具有比迄今为止获得的更好衍射质量的RNase P RNA和复合物的晶体。这正在使用遗传上不同的天然RNA和组分及其突变衍生物来解决。对于SAXS研究，我们与斯克里普斯研究所和劳伦斯伯克利国家实验室的专家合作，早期结果表明这是一种富有成效的方法。公共卫生相关性：该计划将进一步加深我们对不寻常的RNA酶RNase P的理解。RNase P是普遍分布的，是所有细胞生长和发育所必需的，并且可能是生物体特异性抗生素治疗的靶点。

DESCRIPTION (provided by applicant): The universal enzyme ribonuclease P (RNase P) carries out maturation of transfer RNAs (tRNAs) by cleavage of precursor-tRNAs. RNase P is an unusual enzyme in that it is a ribonucleoprotein (RNP) and its catalytic component is comprised of RNA instead of the usual protein. RNA-RNA and RNA-protein interactions are critical to cellular activities, so RNase P offers a specific example of molecular structure and function with broader implications to other cellular functions. The overall goal of this proposal is to determine the structure of bacterial RNase P RNA (~400 nucleotides) and its complexes with RNase P protein (~120 amino acids) and tRNA substrate (~80 nt). Knowledge of these structures is critical to progress in RNase P research. This laboratory has long worked toward this goal, with considerable success, first with biochemical methods and most recently with determination of the crystal structure of the RNase P RNA from Bacillus stearothermophilus at 3.3? and a designed, rapidly crystallizing RNA at 3.6?. However, parts of the crystal structures are distorted or not resolved, and the specific structures of the holoenzyme and ternary complex with tRNA are not known. Even the location of the active site is questionable. At the current stage of resolution, mechanistically relevant properties such as metals, H-bonded networks and waters are not discerned. Moreover, little is known about the solution structure, an important consideration because of potential distortion and disorder in any crystal structures. To address these and other questions we propose to continue crystallographic studies underway to determine molecular structures pertinent to RNase P. To complement, test and refine structure models, small angle X-ray scattering (SAXS) is being used to determine solution structures for comparisons. SAXS results coupled with available crystal structure information will permit modeling the global solution envelope to ~10?, Crystallographic efforts include ongoing analysis of recent data sets, and screens for crystals of RNase P RNA and complexes with better diffraction quality than so far obtained. This is being approached using phylogenetically diverse native RNAs and components, and mutant derivatives thereof. For SAXS studies, we have partnered with experts at Scripps Research Institute and Lawrence Berkeley National Laboratory, and early results show this is a fruitful approach. PUBLIC HEALTH RELEVANCE: The proposed program will further our understanding of the unusual RNA enzyme RNase P. RNase P is universally distributed, required for all cellular growth and development, and potentially is a target for organism-specific antibiotic therapies.