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

拟议的研究涉及生物化学，遗传和结构研究细胞蛋白质如何协助 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， 在体内进行有效的剪接，以帮助RNA折叠成具有催化活性的RNA结构。 在粗糙脉孢菌中，细胞核编码的线粒体酪氨酰-tRNA合成酶（CYT-18）和DEAD-盒 蛋白（CYT-19）是体内剪接三个线粒体I组内含子所必需的，并且这种剪接可以是 用纯化的成分在体外重现。在拟议的研究中，该模型系统将用于 研究DEAD盒蛋白如何被募集并介导RNA构象变化， RNP底物。由于死亡盒蛋白参与了几乎所有涉及RNA的细胞过程， 本研究的结果将广泛适用于了解其他DEAD-box的作用模式 proteins.具体目的是：（1）研究CYT-19、CYT-18和内含子RNA的相互作用。(2)到 确定CYT-19负责靶向CYT-18依赖性I组内含子的区域和CYT-19的区域， CYT-18与CYT-19相互作用。(3)用X射线晶体学方法测定CYT-19的结构。

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.