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

基本信息

批准号：
7613599
负责人：
Nicholas J Reiter
金额：
$ 5.01万
依托单位：
NORTHWESTERN UNIVERSITY
依托单位国家：
美国
项目类别：
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/7613599
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

项目摘要

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，并且还与各种其他 RNA（包括病毒和噬菌体 RNA、mRNA、非编码 RNA）相互作用。 RNA、rRNA 和核糖开关）。在真核生物中，RNase P 成分还充当加工体中 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 生物学功能的出现，了解 RNase P 识别和催化 RNA 的结构基础至关重要。本研究的目的是定义与前体 tRNA 结合的 RNase P 复合物，并了解 RNA/RNA 的作用机制。原子分辨率的 RNA 识别。 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组合在一起。我假设特定 P RNA 区域内存在的高度序列保守性反映了强烈的结构限制，影响 RNA 折叠、底物识别和催化。该项目的目标是：1) 定义全酶 RNase P 与前体 tRNA 底物结合的约 150 kDa 三元复合物的三维结构，2) 确定普遍保守的 RNase P 区域如何参与 RNA 识别和催化。将利用晶体学、生物物理方法和分子生物学技术来表征 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，还可用于识别活性位点内的金属离子。公共健康相关性：本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 RNA 加工引起的人类疾病产生相关影响。 RNase P 特异性的改变已被证明可以有效降解引起疾病的 mRNA，用于治疗白血病（慢性粒细胞性白血病）和病毒，例如单纯疱疹病毒和巨细胞病毒。此外，RNA 介导的 RNase P 在分子水平上的催化作用将有助于深入了解与癌症 (>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，这将极大地帮助 RNA 靶向化疗药物策略的开发。