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

描述(申请人提供)：拟议的研究涉及生化、遗传和结构研究，研究细胞蛋白质如何协助I组内含子的折叠和剪接。第一组内含子是催化的RNA，它在体外自我剪接，但需要蛋白质在体内进行有效剪接，以帮助RNA折叠成催化活性的RNA结构。在粗枝脉孢菌中，线粒体酪氨酰-tRNA合成酶(CyT-18)和DEAD-box蛋白(CyT-19)在体内需要一个核编码的线粒体酪氨酸氨基-tRNA合成酶(CyT-18)和一个DEAD-box蛋白(CyT-19)来剪接三个线粒体I组内含子，并且这种剪接可以用纯化的成分在体外重现。在拟议的研究中，这个模型系统将被用来研究DEAD-BOX蛋白如何被招募到天然RNP底物上并介导RNA构象变化。由于DEAD-box蛋白几乎参与了所有涉及RNA的细胞过程，因此本研究结果将广泛适用于理解其他DEAD-box蛋白的作用方式。具体目的是：(1)研究细胞色素T-19与细胞色素T-18和内含子RNA的相互作用。(2)确定Cyt-19负责靶向Cyt-18依赖的第一组内含子的区域以及Cyt-18与Cyt-19相互作用的区域。(3)用X-射线单晶衍射法确定CyT-19的结构。

DESCRIPTION (provided by applicant): 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 require 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.