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：
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
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/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

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）是负责在蛋白质合成之前生成成熟TRNA的必需核糖核蛋白（RNP）酶，并且还与其他各种RNA相互作用（包括病毒和噬菌体RNA，MRNA，mRNA，非编码RNA，RNA，RRNA，RRNA和核能）。在真核生物中，RNase P成分在加工体中还充当mRNA的协调剂，并通过与非编码RNA基因的染色质结合来调节转录。作为RNase P的生物学功能，重要的是要了解RNase P的RNA识别和催化的结构基础。拟议的研究的目的是用绑定的前体TRNA定义RNase P复合物，并了解原子分辨率下RNA/RNA识别的机理。 P RNA，几个TRNA和各种RNase P蛋白的各个结构是已知的，但尚不清楚这些成分如何在大分子的环境中融合在一起。我假设在特定的P RNA区域内存在的高序列保护是强大结构约束的反映，影响了RNA折叠，底物识别和催化。该项目的目的是：1）定义〜150 kDa三维三维结构的全酶RNase P具有结合的前体TRNA底物，而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用于表征RNase P/tRNA复合物的结构和识别元素。为了获得原子水平的衍射，将实施优化筛选和RNA重组策略。重金属衍生物不仅将用于结构确定，还将用于识别活性位点中的金属离子。公共卫生相关性：本提案中详细介绍的工作将提供有关结构化RNA分子如何相互认识的重要线索，并且对归因于RNA处理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降解疾病，从而导致白血病（慢性髓性白血病）和病毒，例如单纯疱疹病毒和巨细胞病毒。另外，RNA介导的RNase P在分子水平上催化将洞悉与癌症（> 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，从而极大地有助于开发RNA靶向化学治疗药物策略。