Structure and Function of an RNase P ribonucleoprotein-tRNA ternary complex

描述（由申请人提供）：核糖核酸酶P （RNase P）是必需的核糖核蛋白（RNP）酶，负责在蛋白质合成之前生成成熟的trna，并与各种其他RNA（包括病毒和噬菌体RNA、mRNA、非编码RNA、rRNA和核糖开关）相互作用。在真核生物中，RNase P组分还在加工体中充当mRNA的协调者，并通过与非编码RNA基因的染色质结合来调节转录。随着RNase P生物学功能的出现，了解RNase P对RNA识别和催化的结构基础变得至关重要。本研究的目的是定义RNase P复合物与结合前体tRNA，并在原子分辨率上了解RNA/RNA识别的机制。P rna、几种trna和各种RNase P蛋白的个体结构是已知的，但尚不清楚这些成分如何在大分子背景下结合在一起。我假设在特定的P RNA区域中存在的高序列保守性是强结构约束的反映，影响RNA折叠、底物识别和催化。该项目的目的是：1)确定全酶RNase P与结合前体tRNA底物的约150 kDa三元配合物的三维结构；2)确定普遍保守的RNase P区域如何参与RNA识别和催化。晶体学、生物物理方法和分子生物学技术将被用于表征RNase P/tRNA复合物的结构和识别元件。为了获得原子级衍射，将实施优化筛选和RNA重组策略。重金属衍生化不仅可用于结构测定，还可用于鉴定活性位点内的金属离子。公共卫生相关性：本提案中详细介绍的工作将为结构化RNA分子如何相互识别提供重要线索，并对归因于RNA加工的人类疾病具有相关意义。RNase P特异性的改变已被证明可以有效地降解引起疾病的mrna，用于治疗白血病（慢性骨髓性白血病）和病毒，如单纯疱疹病毒和巨细胞病毒。此外，RNase P在分子水平上介导的RNA催化作用将有助于深入了解与癌症（bbb15）和神经退行性疾病相关的RNA加工机制。最后，确定RNase P- tRNA界面将提供重要的结构信息，这将极大地帮助开发RNA靶向化疗药物策略。

DESCRIPTION (provided by applicant): Ribonuclease P (RNase P) is the essential ribonucleoprotein (RNP) enzyme responsible for generating mature tRNAs prior to protein synthesis, and also interacts with various other RNAs (including viral and phage RNA, mRNA, non-coding RNA, rRNA, and riboswitches). In eukaryotes, RNase P components additionally function as coordinators of mRNA in processing bodies, and regulate transcription by binding to the chromatin of non-coding RNA genes. As biological functions of RNase P emerge, it is critical to understand the structural basis of RNA recognition and catalysis by RNase P. The objective of the proposed research is to define the RNase P complex with bound precursor tRNA and to understand the mechanism of RNA/RNA recognition at atomic resolution. Individual structures of P RNAs, several tRNAs, and various RNase P proteins are known, but it is unclear how these components fit together within a macromolecular context. I hypothesize that high sequence conservation present within specific P RNA regions is a reflection of strong structural constraints, affecting RNA folding, substrate recognition, and catalysis. The aims of this project are: 1) to define the three-dimensional structure of a ~150 kDa ternary complex of holoenzyme RNase P with bound precursor tRNA substrate, and 2) to ascertain how universally conserved RNase P regions participate in RNA recognition and catalysis. Crystallography, biophysical methods, and molecular biology techniques will be utilized to characterize the structure and recognition elements of the RNase P/tRNA complex. To obtain atomic level diffraction, optimization screening and RNA recombinant strategies will be implemented. Heavy metal derivatization will be used not only in structure determination, but also to identify metal ions within the active site. PUBLIC HEALTH RELEVANCE: Work detailed in this proposal will provide important clues on how structured RNA molecules recognize each other, and has relevant implications for human diseases attributed to RNA processing. Alteration of RNase P specificity has been shown to efficiently degrade disease causing mRNAs for the treatment of leukemia (chronic myelogenous leukemia) and viruses, such as such as herpes simplex virus and cytomegalovirus. In addition, RNA-mediated catalysis by RNase P at the molecular level will give insight into RNA processing mechanisms associated with cancers (>15) and neurodegenerative diseases. Lastly, defining the RNase P- tRNA interface will provide important structural information that will greatly assist the development of RNA targeted chemotherapeutic drug strategies.