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

描述(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产生成熟的tRNA，也与各种其他RNA(包括病毒和噬菌体RNA、信使核糖核酸、非编码RNA、rRNA和核糖开关)相互作用。在真核生物中，核糖核酸酶P组分还作为加工体中的mRNA的协调者，通过与非编码RNA基因的染色质结合来调节转录。随着RNaseP生物学功能的出现，了解RNaseP识别和催化RNA的结构基础变得至关重要。本研究的目的是定义RNaseP与结合前体tRNA的复合体，并在原子分辨率上了解RNA/RNA识别的机制。已知P RNAs、几个tRNAs和各种RNaseP蛋白的单独结构，但尚不清楚这些成分如何在大分子环境中结合在一起。我假设，特定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。本项目的目标是：1)确定全酶RNaseP与结合前体tRNA底物的~150 kDa三元复合体的三维结构；2)确定普遍保守的RNase P区如何参与RNA识别和催化。将利用结晶学、生物物理方法和分子生物学技术来表征RNaseP/tRNA复合体的结构和识别元件。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，还可用于鉴定活性中心内的金属离子。公共卫生相关性：本提案中详细介绍的工作将提供关于结构RNA分子如何相互识别的重要线索，并对可归因于RNA加工的人类疾病具有相关影响。RNase P特异性的改变已被证明能有效地降解引起疾病的mRNAs，用于治疗白血病(慢性粒细胞白血病)和病毒，如单纯疱疹病毒和巨细胞病毒。此外，RNase P在分子水平上的RNA介导的催化作用将使人们深入了解与癌症(&gt；15)和神经退行性疾病相关的RNA处理机制。最后，定义RNaseP-tRNA界面将提供重要的结构信息，这将极大地帮助RNA靶向化疗药物策略的发展。

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.