Structure and function of a DEAD-box protein in group I intron splicing

I组内含子剪接中DEAD-box蛋白的结构和功能

基本信息

批准号：
7318347
负责人：
MARK DEL CAMPO
金额：
$ 5.04万
依托单位：
UNIVERSITY OF TEXAS AT AUSTIN
依托单位国家：
美国
项目类别：
财政年份：
2005
资助国家：
美国
起止时间：
2005-12-02 至 2008-12-01
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7318347
关键词：
ATP phosphohydrolase Active Sites Alleles Amino Acids Bacillus subtilis yxiN protein Binding Biochemical Genetics Biogenesis Biological Assay Biological Models Boxing C-terminal Catalysis Catalytic RNA Cell physiology Chemical Structure Complex Crystallization Defect Disruption Escherichia coli Family Fellowship Gel Chromatography Genetic Guanosine Helix (Snails)Heterogeneous Nuclear RNA Homologous Gene Immunoblotting In Vitro Individual Introns Kinetics Mediating Mitochondria Modeling Molecular Chaperones Mutagenesis Mutation N-terminal Names Neurospora crassa Nuclear Numbers Organism Peripheral Precursor RNA Protein Overexpression Proteins RNA RNA Folding RNA Helicase RNA Splicing RNA-Binding Proteins Reaction Recruitment Activity Research Ribosomal RNA Ribosomes Saccharomyces cerevisiae Specificity Structure Testing Tetrahymena Tyrosine-tRNA Ligase Viral X-Ray Crystallography cancer therapy cancer type cofactor ear helix helicase in vivo mRNA Precursor mutant nucleotide binding fold protein function protein protein interaction three dimensional structure

项目摘要

The proposed research involves biochemical, genetic, and structural studies of how cellular proteins assist in the folding and splicing of group I introns. Group I introns are catalytic RNAs that self-splice in vitro but rerquire proteins for efficient splicing in vivo, to help the RNA fold into the catalytically active RNA structure. In Neurospora crassa, a nuclear-encoded mitochondrial tyrosyl-tRNA synthetase (CYT-18) and a DEAD-box protein (CYT-19) are required for splicing three mitochondrial group I introns in vivo, and this splicing can be recapitulated in vitro with purified components. In the proposed research, this model system will be used to investigate how DEAD-box proteins are recruited to and mediate RNA conformational changes on a natural RNP substrate. Since DEAD-box proteins are involved in virtually all cellular processes involving RNA, results of this study will be widely applicable to understanding the mode of action of other DEAD-box proteins. Specific aims are: (1) To investigate the interaction of CYT-19 with CYT-18 and intron RNA. (2) To identify the regions of CYT-19 responsible for targeting CYT-18-dependent group I introns and the regions of CYT-18 interacting with CYT-19. (3) To determine the structure of CYT-19 by X-ray crystallography.

拟议的研究涉及细胞蛋白质如何协助的生物化学、遗传和结构研究 I 组内含子的折叠和剪接。 I 组内含子是在体外自我剪接的催化 RNA，但需要蛋白质在体内进行有效剪接，以帮助 RNA 折叠成具有催化活性的 RNA 结构。在粗糙脉孢菌中，核编码的线粒体酪氨酰-tRNA 合成酶 (CYT-18) 和 DEAD-box 体内剪接三个线粒体 I 组内含子需要蛋白质（CYT-19），并且这种剪接可以是用纯化的成分在体外进行重现。在拟议的研究中，该模型系统将用于研究 DEAD-box 蛋白如何招募并介导自然RNA构象变化 RNP 底物。由于 DEAD-box 蛋白几乎参与所有涉及 RNA 的细胞过程，这项研究的结果将广泛应用于理解其他 DEAD-box 的作用模式蛋白质。具体目的是：（1）研究CYT-19与CYT-18和内含子RNA的相互作用。 (2) 至确定负责靶向 CYT-18 依赖性 I 组内含子的 CYT-19 区域和 CYT-18 与 CYT-19 相互作用。 (3)通过X射线晶体学确定CYT-19的结构。