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

RNase P 核糖核蛋白-tRNA 三元复合物的结构和功能

• 批准号: 7769551
• 负责人: Nicholas J Reiter
• 金额: $ 5.22万
• 依托单位: NORTHWESTERN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-01-01 至 2011-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7769551
• 关键词: Active Sites; Affect; Binding; Biological Process; Catalysis; Chromatin; Chronic Myeloid Leukemia; Complex; Crystallography; Cytomegalovirus; Development; Disease; Disease Attributes; Elements; Enzymes; Eukaryota; Functional RNA; Genes; Genetic Transcription; Glycine decarboxylase; Heavy Metals; Holoenzymes; Individual; Ions; Malignant Neoplasms; Mediating; Messenger RNA; Metals; Methods; Molecular; Molecular Biology Techniques; Neurodegenerative Disorders; Nucleotides; Pharmaceutical Preparations; Process; Protein Biosynthesis; Proteins; RNA; RNA Folding; RNA I; RNA Phages; RNA Precursors; RNA Processing; RNase P; Research; Resolution; Ribonucleoproteins; Ribosomal RNA; Screening procedure; Simplexvirus; Site-Directed Mutagenesis; Specificity; Structure; Transfer RNA; Viral; Work; base; design; human disease; insight; leukemia; leukemia virus; public health relevance; recombinant RNA; stoichiometry; tRNA Precursor; three dimensional structure

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.

描述（由申请人提供）：核糖核酸酶P（RNase P）是负责在蛋白质合成前产生成熟tRNA的必需核糖核蛋白（RNP）酶，也与各种其他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组分还在加工体中作为mRNA的协调子，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的生物学功能的出现，理解RNase P识别和催化RNA的结构基础是至关重要的。拟议研究的目的是定义RNase P与结合的前体tRNA的复合物，并理解原子分辨率下RNA/RNA识别的机制。P RNA、几种tRNA和各种RNase P蛋白的单个结构是已知的，但尚不清楚这些组分如何在大分子背景下结合在一起。我推测，高序列保守性存在于特定的P RNA区域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与结合的前体tRNA底物的~150 kDa三元复合物的三维结构，以及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、生物物理学方法和分子生物学技术将用于表征RNase P/tRNA复合物的结构和识别元件。为了获得原子水平的衍射，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，还可用于鉴定活性部位内的金属离子。公共卫生相关性：该提案中详细的工作将为结构化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。已经显示RNase P特异性的改变有效地降解致病mRNA，用于治疗白血病（慢性髓性白血病）和病毒，例如单纯疱疹病毒和巨细胞病毒。此外，RNA酶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（> 15）和神经退行性疾病相关的RNA加工机制。最后，定义RNase P-tRNA界面将提供重要的结构信息，这将极大地有助于RNA靶向化疗药物策略的开发。